U.S. patent application number 13/849342 was filed with the patent office on 2013-10-10 for antibodies and diagnostics.
The applicant listed for this patent is UCB Pharma S.A.. Invention is credited to John Latham, David G. Winkler.
Application Number | 20130267689 13/849342 |
Document ID | / |
Family ID | 39865706 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130267689 |
Kind Code |
A1 |
Latham; John ; et
al. |
October 10, 2013 |
ANTIBODIES AND DIAGNOSTICS
Abstract
Compositions and methods relating to sclerostin binding agents,
such as antibodies and polypeptides capable of binding to
sclerostin, are provided.
Inventors: |
Latham; John; (Seattle,
WA) ; Winkler; David G.; (Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB Pharma S.A. |
Slough |
|
GB |
|
|
Family ID: |
39865706 |
Appl. No.: |
13/849342 |
Filed: |
March 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12513806 |
May 6, 2009 |
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PCT/US2007/084280 |
Nov 9, 2007 |
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13849342 |
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60857870 |
Nov 10, 2006 |
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Current U.S.
Class: |
530/389.8 ;
435/252.33; 435/320.1; 435/332; 435/69.6; 536/23.53 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/22 20130101; C07K 2317/56 20130101; C07K 2317/565
20130101 |
Class at
Publication: |
530/389.8 ;
536/23.53; 435/320.1; 435/69.6; 435/332; 435/252.33 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1.-35. (canceled)
36. A polypeptide that cross-blocks the binding of at least one of
antibodies Antibody A--Antibody Y to sclerostin.
37. The polypeptide of claim 36, comprising one or more
complementarity determining regions of any one of Antibody
A--Antibody Y, wherein the polypeptide comprises a binding affinity
for human sclerostin of less than or equal to
1.times.10.sup.-7M.
38. The polypeptide of claim 37, comprising three or more CDRs of
any one of Antibody A--Antibody Y.
39. The polypeptide of claim 38, comprising six CDRs of any one of
Antibody A--Antibody Y.
40. The polypeptide of claim 37, wherein the polypeptide comprises
one or more CDR amino acid sequences selected from the group
consisting of SEQ ID NOs: 109, 110, 111, 115, 116, 117, 121, 122,
123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147,
151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175,
176, 177, 181, 182, 183, 187, 188, 189, 193, 194, 195, 199, 200,
201, 205, 206, 207, 211, 212, 213, 217, 218, 219, 223, 224, 225,
229, 230, 231, 235, 236, 237, 241, 242, 243, 247, 248, 249, 253,
254, and 255.
41. The polypeptide of claim 37, wherein the polypeptide comprises
one or more CDR amino acid sequences selected from the group
consisting of SEQ ID NOs: 106, 107, 108, 112, 113, 114, 118, 119,
120, 124, 125, 126, 130, 131, 132, 136, 137, 138, 142, 143, 144,
148, 149, 150, 154, 155, 156, 160, 161, 162, 166, 167, 168, 172,
173, 174, 178, 179, 180, 184, 185, 186, 190, 191, 192, 196, 197,
198, 202, 203, 204, 208, 209, 210, 214, 215, 216, 220, 221, 222,
226, 227, 228, 232, 233, 234, 238, 239, 240, 244, 245, 246, 250,
251, and 252.
42. The polypeptide of claim 38, comprising: (a) CDR sequences of
SEQ ID NOs: 106, 107, and 108; (b) CDR sequences of SEQ ID NOs:
109, 110, and 111; (c) CDR sequences of SEQ ID NOs: 112, 113, and
114; (d) CDR sequences of SEQ ID NOs: 115, 116, and 117; (e) CDR
sequences of SEQ ID NOs: 118, 119, and 120; (f) CDR sequences of
SEQ ID NOs: 121, 122, and 123; (g) CDR sequences of SEQ ID NOs:
124, 125, and 126; (h) CDR sequences of SEQ ID NOs: 127, 128, and
129; (i) CDR sequences of SEQ ID NOs: 130, 131, and 132; (j) CDR
sequences of SEQ ID NOs: 133, 134, and 135; (k) CDR sequences of
SEQ ID NOs: 136, 137, and 138; (l) CDR sequences of SEQ ID NOs:
139, 140, and 141; (m) CDR sequences of SEQ ID NOs: 142, 143, and
144; (n) CDR sequences of SEQ ID NOs: 145, 146, and 147; (o) CDR
sequences of SEQ ID NOs: 148, 149, and 150; (p) CDR sequences of
SEQ ID NOs: 151, 152, and 153; (q) CDR sequences of SEQ ID NOs:
154, 155, and 156; (r) CDR sequences of SEQ ID NOs: 157, 158, and
159; (s) CDR sequences of SEQ ID NOs: 160, 161, and 162; (t) CDR
sequences of SEQ ID NOs: 163, 164, and 165; (u) CDR sequences of
SEQ ID NOs: 166, 167, and 168; (v) CDR sequences of SEQ ID NOs:
169, 170, and 171; (w) CDR sequences of SEQ ID NOs: 172, 173, and
174; (x) CDR sequences of SEQ ID NOs: 175, 176, and 177; (y) CDR
sequences of SEQ ID NOs: 178, 179, and 180; (z) CDR sequences of
SEQ ID NOs: 181, 182, and 183; (aa) CDR sequences of SEQ ID NOs:
184, 185, and 186; (bb) CDR sequences of SEQ ID NOs: 187, 188, and
189; (cc) CDR sequences of SEQ ID NOs: 190, 191, and 192; (dd) CDR
sequences of SEQ ID NOs: 193, 194, and 195; (ee) CDR sequences of
SEQ ID NOs: 196, 197, and 198; (ft) CDR sequences of SEQ ID NOs:
199, 200, and 201; (gg) CDR sequences of SEQ ID NOs: 202, 203, and
204; (hh) CDR sequences of SEQ ID NOs: 205, 206, and 207; (ii) CDR
sequences of SEQ ID NOs: 208, 209, and 210; (jj) CDR sequences of
SEQ ID NOs: 211, 212, and 213; (kk) CDR sequences of SEQ ID NOs:
214, 215, and 216; (ll) CDR sequences of SEQ ID NOs: 217, 218, and
219; (mm) CDR sequences of SEQ ID NOs: 220, 221, and 222; (nn) CDR
sequences of SEQ ID NOs: 223, 224, and 225; (oo) CDR sequences of
SEQ ID NOs: 226, 227, and 228; (pp) CDR sequences of SEQ ID NOs:
229, 230, and 231; (qq) CDR sequences of SEQ ID NOs: 232, 233, and
234; (rr) CDR sequences of SEQ ID NOs: 235, 236, and 237; (ss) CDR
sequences of SEQ ID NOs: 238, 239, and 240; (tt) CDR sequences of
SEQ ID NOs: 241, 242, and 243; (uu) CDR sequences of SEQ ID NOs:
244, 245, and 246; (vv) CDR sequences of SEQ ID NOs: 247, 248, and
249; (ww) CDR sequences of SEQ ID NOs: 250, 251, and 252; or (xx)
CDR sequences of SEQ ID NOs: 253, 254, and 255.
43. The polypeptide of claim 42, comprising: (a) CDR sequences of
SEQ ID NOs: 106, 107, and 108 and CDR sequences of SEQ ID NOs: 109,
110, and 111; (b) CDR sequences of SEQ ID NOs: 112, 113, and 114
and CDR sequences of SEQ ID NOs: 115, 116, and 117; (c) CDR
sequences of SEQ ID NOs: 118, 119, and 120 and CDR sequences of SEQ
ID NOs: 121, 122, and 123; (d) CDR sequences of SEQ ID NOs: 124,
125, and 126 and CDR sequences of SEQ ID NOs: 127, 128, and 129;
(e) CDR sequences of SEQ ID NOs: 130, 131, and 132 and CDR
sequences of SEQ ID NOs: 133, 134, and 135; (f) CDR sequences of
SEQ ID NOs: 136, 137, and 138 and CDR sequences of SEQ ID NOs: 139,
140, and 141; (g) CDR sequences of SEQ ID NOs: 142, 143, and 144
and CDR sequences of SEQ ID NOs: 145, 146, and 147; (h) CDR
sequences of SEQ ID NOs: 148, 149, and 150 and CDR sequences of SEQ
ID NOs: 151, 152, and 153; (i) CDR sequences of SEQ ID NOs: 154,
155, and 156 and CDR sequences of SEQ ID NOs: 157, 158, and 159;
(j) CDR sequences of SEQ ID NOs: 160, 161, and 162 and CDR
sequences of SEQ ID NOs: 163, 164, and 165; (k) CDR sequences of
SEQ ID NOs: 166, 167, and 168 and CDR sequences of SEQ ID NOs: 169,
170, and 171; (l) CDR sequences of SEQ ID NOs: 172, 173, and 174
and CDR sequences of SEQ ID NOs: 175, 176, and 177; (m) CDR
sequences of SEQ ID NOs: 178, 179, and 180 and CDR sequences of SEQ
ID NOs: 181, 182, and 183; (n) CDR sequences of SEQ ID NOs: 184,
185, and 186 and CDR sequences of SEQ ID NOs: 187, 188, and 189;
(o) CDR sequences of SEQ ID NOs: 190, 191, and 192 and CDR
sequences of SEQ ID NOs: 193, 194, and 195; (p) CDR sequences of
SEQ ID NOs: 196, 197, and 198 and CDR sequences of SEQ ID NOs: 199,
200, and 201; (q) CDR sequences of SEQ ID NOs: 202, 203, and 204
and CDR sequences of SEQ ID NOs: 205, 206, and 207; (r) CDR
sequences of SEQ ID NOs: 208, 209, and 210 and CDR sequences of SEQ
ID NOs: 211, 212, and 213; (s) CDR sequences of SEQ ID NOs: 214,
215, and 216 and CDR sequences of SEQ ID NOs: 217, 218, and 219;
(t) CDR sequences of SEQ ID NOs: 220, 221, and 222 and CDR
sequences of SEQ ID NOs: 223, 224, and 225; (u) CDR sequences of
SEQ ID NOs: 226, 227, and 228 and CDR sequences of SEQ ID NOs: 229,
230, and 231; (v) CDR sequences of SEQ ID NOs: 232, 233, and 234
and CDR sequences of SEQ ID NOs: 235, 236, and 237; (w) CDR
sequences of SEQ ID NOs: 238, 239, and 240 and CDR sequences of SEQ
ID NOs: 241, 242, and 243; (x) CDR sequences of SEQ ID NOs: 244,
245, and 246 and CDR sequences of SEQ ID NOs: 247, 248, and 249; or
(y) CDR sequences of SEQ ID NOs: 250, 251, and 252 and CDR
sequences of SEQ ID NOs: 253, 254, and 255.
44. The polypeptide of claim 37, comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18,
22, 26, 30, 34, 38, 42, 46, 50, 56, 60, 64, 68, 72, 76, 80, 84, 88,
92, 96, and 100.
45. The polypeptide of claim 37, comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20,
24, 28, 32, 36, 40, 44, 48, 52, 58, 62, 66, 70, 74, 78, 82, 86, 90,
94, 98, and 102.
46. The polypeptide of claim 37, wherein the polypeptide comprises
a binding affinity for human sclerostin of less than or equal to
1.times.10.sup.-8 M.
47. The polypeptide of claim 43, wherein the polypeptide comprises
a binding affinity for human sclerostin of less than or equal to
1.times.10.sup.-9 M.
48. An isolated nucleic acid molecule comprising a polynucleotide
encoding the polypeptide of claim 37.
49. A vector comprising the isolated nucleic acid molecule of claim
48.
50. A host cell comprising the vector of claim 49.
51. A method of making a protein comprising culturing a host cell
of claim 50 under conditions wherein the encoded protein is
expressed.
52. An isolated antibody selected from the group consisting of
Antibody A--Antibody Y.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to epitopes of
sclerostin protein, including human sclerostin protein, and binding
agents (such as antibodies) capable of binding to sclerostin or
fragments thereof.
BACKGROUND OF THE INVENTION
[0002] Two or three distinct phases of changes to bone mass occur
over the life of an individual (see Riggs, West J. Med., 154:63-77
(1991)). The first phase occurs in both men and women and proceeds
to attainment of a peak bone mass. This first phase is achieved
through linear growth of the endochondral growth plates and radial
growth due to a rate of periosteal apposition. The second phase
begins around age 30 for trabecular bone (flat bones such as the
vertebrae and pelvis) and about age 40 for cortical bone (e.g.,
long bones found in the limbs) and continues to old age. This phase
is characterized by slow bone loss and occurs in both men and
women. In women, a third phase of bone loss also occurs, most
likely due to postmenopausal estrogen deficiencies. During this
phase alone, women may lose an additional bone mass from the
cortical bone and from the trabecular compartment (see Riggs,
supra).
[0003] Loss of bone mineral content can be caused by a wide variety
of conditions and may result in significant medical problems. For
example, osteoporosis is a debilitating disease in humans and is
characterized by marked decreases in skeletal bone mass and mineral
density, structural deterioration of bone, including degradation of
bone microarchitecture and corresponding increases in bone
fragility (i.e., decreases in bone strength), and susceptibility to
fracture in afflicted individuals. Osteoporosis in humans is
generally preceded by clinical osteopenia (bone mineral density
that is greater than one standard deviation but less than 2.5
standard deviations below the mean value for young adult bone), a
condition found in approximately 25 million people in the United
States. Another 7-8 million patients in the United States have been
diagnosed with clinical osteoporosis (defined as bone mineral
content greater than 2.5 standard deviations below that of mature
young adult bone). The frequency of osteoporosis in the human
population increases with age. Among Caucasians, osteoporosis is
predominant in women who, in the United States, comprise 80% of the
osteoporosis patient pool. The increased fragility and
susceptibility to fracture of skeletal bone in the aged is
aggravated by the greater risk of accidental falls in this
population. Fractured hips, wrists, and vertebrae are among the
most common injuries associated with osteoporosis. Hip fractures in
particular are extremely uncomfortable and expensive for the
patient, and for women, correlate with high rates of mortality and
morbidity.
[0004] Although osteoporosis has been regarded as an increase in
the risk of fracture due to decreased bone mass, few of the
presently available treatments for skeletal disorders can increase
the bone density of adults, and most of the presently available
treatments work primarily by inhibiting further bone resorption
rather than stimulating new bone formation. Estrogen is now being
prescribed to retard bone loss. However, some controversy exists
over whether patients gain any long-term benefit and whether
estrogen has any effect on patients over 75 years old. Moreover,
use of estrogen is believed to increase the risk of breast and
endometrial cancer. Calcitonin, osteocalcin with vitamin K, or high
doses of dietary calcium, with or without vitamin D, have also been
suggested for postmenopausal women. High doses of calcium, however,
often have undesired gastrointestinal side effects, and serum and
urinary calcium levels must be continuously monitored (e.g., Khosla
and Riggs, Mayo Clin. Proc., 70:978982 (1995)).
[0005] Other current therapeutic approaches to osteoporosis include
bisphosphonates (e.g., Fosamax.TM., Actonel.TM., Bonviva.TM.,
Zometa.TM., olpadronate, neridronate, skelid, bonefos), parathyroid
hormone, calcilytics, calcimimetics (e.g., cinacalcet), statins,
anabolic steroids, lanthanum and strontium salts, and sodium
fluoride. Such therapeutics, however, are often associated with
undesirable side effects (see Khosla and Riggs, supra).
[0006] Sclerostin, the product of the SOST gene, is absent in
sclerosteosis, a skeletal disease characterized by bone overgrowth
and strong dense bones (Brunkow et al., Am. J. Hum. Genet.,
68:577-589 (2001); Balemans et al., Hum. Mol. Genet., 10:537-543
(2001)). The amino acid sequence of human sclerostin is reported by
Brunkow et al. ibid and is disclosed herein as SEQ ID NO:1.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention relates to an isolated antibody selected from
the group consisting of antibodies A, B, C, D, E, F, G, H, I, J, K,
L, M, N, O, P, Q, R, S, T, U, V, W, X, and Y; the isolated
antibody, or an antigen-binding fragment thereof, may be a
polyclonal antibody, a monoclonal antibody, a humanized antibody, a
human antibody, or a human/non-human chimeric antibody, such as a
mouse/human or rabbit/human chimeric antibody.
[0008] The invention further relates to a methods for detecting,
diagnosing, and determining the progression or regression of a bone
disorder associated with at least one of low bone mass, low bone
mineral density, and poor bone quality in a mammalian subject which
comprises obtaining a biological sample from a subject suspected of
suffering from the disorder, contacting the biological sample with
an agent capable of detecting sclerostin, and identifying or
quantitating a binding complex between the agent and sclerostin,
wherein the agent comprises an anti-sclerostin antibody, or
sclerostin-binding fragment thereof.
[0009] Provided herein are antibodies that specifically bind to
human sclerostin. The antibodies can be characterized by their
ability to bind to human sclerostin or a fragment thereof.
[0010] Also provided is an isolated antibody, or an antigen-binding
fragment thereof, that specifically binds to human sclerostin and
has at least one CDR sequence selected from SEQ ID NOs: 106-255 and
variants thereof.
[0011] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entireties as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the amino acid sequence of the mature form
(signal peptide cleaved off) of human sclerostin (SEQ ID NO:1).
Also depicted is the nucleotide sequence of the human sclerostin
coding region that encodes the mature form of human sclerostin. The
eight cysteines are numbered C1 through C8. The cystine-knot is
formed by three disulfide bonds (C1-C5; C3-C7; C4-C8). C2 and C6
also form a disulfide bond, however this disulfide is not part of
the cystine-knot.
[0013] FIG. 2 depicts a schematic of the basic structure of human
sclerostin. There is an N-terminal arm (from the first Q to C1) and
a C-terminal arm (from C8 to the terminal Y). In between these arms
there is the cystine-knot structure (formed by three disulfides:
C1-C5; C3-C7; C4-C8) and three loops which are designated Loop 1,
Loop 2 and Loop 3. The distal regions of Loop 1 and Loop 3 are
linked by the C2-C6 disulfide. Potential trypsin cleavage sites are
indicated (arginine=R and lysine=K). Some of the potential AspN
cleavage sites are indicated [only aspartic acid (D) residues are
shown].
DETAILED DESCRIPTION
[0014] The present invention relates to regions of the human
sclerostin protein that contain epitopes recognized by antibodies
that also bind to full-length sclerostin, and methods of making and
using these epitopes. The invention also provides binding agents
(such as antibodies) that specifically bind to sclerostin or
portions of sclerostin, and methods for using such binding agents.
The binding agents are useful to block or impair binding of human
sclerostin to one or more ligand.
[0015] Recombinant human sclerostin/SOST is commercially available
from R&D Systems (Minneapolis, Minn., USA; 2006
cat#1406-ST-025). Additionally, recombinant mouse sclerostin/SOST
is commercially available from R&D Systems (Minneapolis, Minn.,
USA; 2006 cat#1589-ST-025). Research grade sclerostin binding
monoclonal antibodies are commercially available from R&D
Systems (Minneapolis, Minn., USA; mouse monoclonal: 2006 cat#
MAB1406; rat monoclonal: 2006 cat# MAB1589). U.S. Pat. Nos.
6,395,511 and 6,803,453, and U.S. Patent Publications 2004/0009535
and 2005/0106683 refer to anti-sclerostin antibodies generally.
[0016] As used herein, the term human sclerostin is intended to
include the protein of SEQ ID NO:1 and allelic variants thereof.
Sclerostin can be purified from 293T host cells that have been
transfected by a gene encoding sclerostin by elution of filtered
supernatant of host cell culture fluid using a Heparin HP column,
using a salt gradient. The preparation and further purification
using cation exchange chromatography are described in Examples 1
and 2.
[0017] Binding agents of the invention are preferably antibodies,
as defined herein. The term "antibody" refers to an intact
antibody, or a binding fragment thereof. An antibody may comprise a
complete antibody molecule (including polyclonal, monoclonal,
chimeric, humanized, or human versions having full length heavy
and/or light chains), or comprise an antigen binding fragment
thereof. Antibody fragments include F(ab').sub.2, Fab, Fab', Fv,
Fc, and Fd fragments, and can be incorporated into single domain
antibodies, single-chain antibodies, maxibodies, minibodies,
intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv
(see e.g., Hollinger and Hudson, Nature Biotechnology,
23(9):1126-1136 (2005)). Antibody polypeptides are also disclosed
in U.S. Pat. No. 6,703,199, including fibronectin polypeptide
monobodies. Other antibody polypeptides are disclosed in U.S.
Patent Publication 2005/0238646, which are single-chain
polypeptides.
[0018] Antigen binding fragments derived from an antibody can be
obtained, for example, by proteolytic hydrolysis of the antibody,
for example, pepsin or papain digestion of whole antibodies
according to conventional methods. By way of example, antibody
fragments can be produced by enzymatic cleavage of antibodies with
pepsin to provide a 5S fragment termed F(ab').sub.2. This fragment
can be further cleaved using a thiol reducing agent to produce 3.5S
Fab' monovalent fragments. Optionally, the cleavage reaction can be
performed using a blocking group for the sulfhydryl groups that
result from cleavage of disulfide linkages. As an alternative, an
enzymatic cleavage using papain produces two monovalent Fab
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et
al., Arch. Biochem. Biophys., 89:230 (1960); Porter, Biochem. J.,
73:119 (1959); Edelman et al., in Methods in Enzymology, 1:422
(Academic Press 1967); and by Andrews, S. M. and Titus, J. A. in
Current Protocols in Immunology (Coligan J. E., et al., eds), John
Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and
2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as
separating heavy chains to form monovalent light-heavy chain
fragments (Fd), further cleaving of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0019] An antibody fragment may also be any synthetic or
genetically engineered protein. For example, antibody fragments
include isolated fragments consisting of the light chain variable
region, "Fv" fragments consisting of the variable regions of the
heavy and light chains, and recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker (scFv proteins).
[0020] Another form of an antibody fragment is a peptide comprising
one or more complementarity determining regions (CDRs) of an
antibody. CDRs (also termed "minimal recognition units," or
"hypervariable region") can be obtained by constructing
polynucleotides that encode the CDR of interest. Such
polynucleotides are prepared, for example, by using the polymerase
chain reaction to synthesize the variable region using mRNA of
antibody-producing cells as a template (see, for example, Larrick
et al., Methods: A Companion to Methods in Enzymology 2:106, 1991;
Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in
Monoclonal Antibodies: Production, Engineering and Clinical
Application, Ritter et al. (eds.), page 166 (Cambridge University
Press 1995); and Ward et al., "Genetic Manipulation and Expression
of Antibodies," in Monoclonal Antibodies: Principles and
Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc.
1995)).
[0021] Thus, in one embodiment, the binding agent comprises at
least one CDR as described herein. The binding agent may comprise
at least two, three, four, five or six CDR's as described herein.
The binding agent further may comprise at least one variable region
domain of an antibody described herein. The variable region domain
may be of any size or amino acid composition and will generally
comprise at least one CDR sequence responsible for binding to human
sclerostin, for example CDR-H1, CDR-H2, CDR-H3 and/or the light
chain CDRs specifically described herein and which is adjacent to
or in frame with one or more framework sequences. In general terms,
the variable (V) region domain may be any suitable arrangement of
immunoglobulin heavy (V.sub.H) and/or light (V.sub.L) chain
variable domains. Thus, for example, the V region domain may be
monomeric and be a V.sub.H or V.sub.L domain, which is capable of
independently binding human sclerostin with an affinity at least
equal to 1.times.10.sup.-7M or less as described below.
Alternatively, the V region domain may be dimeric and contain
V.sub.H-V.sub.H, V.sub.H-V.sub.L, or V.sub.L-V.sub.L, dimers. The V
region dimer comprises at least one V.sub.H and at least one
V.sub.L chain that may be non-covalently associated (hereinafter
referred to as F.sub.v). If desired, the chains may be covalently
coupled either directly, for example, via a disulfide bond between
the two variable domains, or through a linker, for example, a
peptide linker, to form a single chain Fv (scF.sub.v).
[0022] The variable region domain may be any naturally occurring
variable domain or an engineered version thereof. By engineered
version is meant a variable region domain that has been created
using recombinant DNA engineering techniques. Such engineered
versions include those created, for example, from a specific
antibody variable region by insertions, deletions, or changes in or
to the amino acid sequences of the specific antibody. Particular
examples include engineered variable region domains containing at
least one CDR and optionally one or more framework amino acids from
a first antibody and the remainder of the variable region domain
from a second antibody.
[0023] The variable region domain may be covalently attached at a
C-terminal amino acid to at least one other antibody domain or a
fragment thereof. Thus, for example, a V.sub.H domain that is
present in the variable region domain may be linked to an
immunoglobulin CH1 domain, or a fragment thereof. Similarly a
V.sub.L domain may be linked to a C.sub.K domain or a fragment
thereof. In this way, for example, the antibody may be an Fab
fragment wherein the antigen binding domain contains associated
V.sub.H and V.sub.L domains covalently linked at their C-termini to
a CH1 and C.sub.K domain, respectively. The CH1 domain may be
extended with further amino acids, for example, to provide a hinge
region or a portion of a hinge region domain as found in a Fab'
fragment, or to provide further domains, such as antibody CH2 and
CH3 domains.
[0024] As described herein, binding agents comprise at least one of
these CDRs. For example, one or more CDR may be incorporated into
known antibody framework regions (IgG1, IgG2, etc.), or conjugated
to a suitable vehicle to enhance the half-life thereof. Suitable
vehicles include, but are not limited to Fc, polyethylene glycol
(PEG), albumin, transferrin, and the like. These and other suitable
vehicles are known in the art. Such conjugated CDR peptides may be
in monomeric, dimeric, tetrameric, or other form. In one
embodiment, one or more water-soluble polymer is bonded at one or
more specific position, for example at the amino terminus, of a
binding agent.
[0025] In certain preferred embodiments, a binding agent comprises
one or more water soluble polymer attachments, including, but not
limited to, polyethylene glycol, polyoxyethylene glycol, or
polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835,
4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. In
certain embodiments, a derivative binding agent comprises one or
more of monomethoxy-polyethylene glycol, dextran, cellulose, or
other carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of such polymers. In certain embodiments, one or more water-soluble
polymers are randomly attached to one or more side chains. In
certain embodiments, PEG can act to improve the therapeutic
capacity for a binding agent, such as an antibody. Certain such
methods are discussed, for example, in U.S. Pat. No. 6,133,426,
which is hereby incorporated by reference for any purpose.
[0026] It will be appreciated that a binding agent of the present
invention may have at least one amino acid substitution, providing
that the binding agent retains binding specificity. Therefore,
modifications to the binding agent structures are encompassed
within the scope of the invention. These may include amino acid
substitutions, which may be conservative or non-conservative, that
do not destroy the sclerostin binding capability of a binding
agent. Conservative amino acid substitutions may encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties. A conservative
amino acid substitution may also involve a substitution of a native
amino acid residue with a normative residue such that there is
little or no effect on the polarity or charge of the amino acid
residue at that position.
[0027] Non-conservative substitutions may involve the exchange of a
member of one class of amino acids or amino acid mimetics for a
member from another class with different physical properties (e.g.,
size, polarity, hydrophobicity, and/or charge). Such substituted
residues may be introduced into regions of the human antibody that
are homologous with non-human antibodies, or into the
non-homologous regions of the molecule.
[0028] Moreover, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. The variants can then be screened using activity
assays known to those skilled in the art. Such variants could be
used to gather information about other suitable variants. For
example, if one discovered that a change to a particular amino acid
residue resulted in destroyed, undesirably reduced, or unsuitable
activity, variants with such a change may be avoided. In other
words, based on information gathered from such routine experiments,
one skilled in the art can readily determine the amino acids where
further substitutions should be avoided either alone or in
combination with other mutations.
[0029] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth herein using well-known
techniques. In certain embodiments, one skilled in the art may
identify suitable areas of the molecule that may be changed without
destroying activity by targeting regions not believed to be
important for activity. In certain embodiments, one can identify
residues and portions of the molecules that are conserved among
similar polypeptides. In certain embodiments, even areas that may
be important for biological activity or for structure may be
subject to conservative amino acid substitutions without destroying
the biological activity or without adversely affecting the
polypeptide structure.
[0030] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a protein that correspond to amino acid residues which
are important for activity or structure in similar proteins. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues.
[0031] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of an antibody with respect to its three
dimensional structure. In certain embodiments, one skilled in the
art may choose not to make radical changes to amino acid residues
predicted to be on the surface of the protein, since such residues
may be involved in important interactions with other molecules.
[0032] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol.,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural database
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will become dramatically more accurate.
[0033] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage"
(See Holm, supra (1999), and Brenner, supra (1997)).
[0034] In certain embodiments, variants of binding agents include
glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of a parent polypeptide. In certain embodiments, variants
comprise a greater or a lesser number of N-linked glycosylation
sites than the native protein. An N-linked glycosylation site is
characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the
amino acid residue designated as X may be any amino acid residue
except proline. The substitution of amino acid residues to create
this sequence provides a potential new site for the addition of an
N-linked carbohydrate chain. Alternatively, substitutions which
eliminate this sequence will remove an existing N-linked
carbohydrate chain. Also provided is a rearrangement of N-linked
carbohydrate chains wherein one or more N-linked glycosylation
sites (typically those that are naturally occurring) are eliminated
and one or more new N-linked sites are created. Additional
preferred antibody variants include cysteine variants wherein one
or more cysteine residues are deleted from or substituted for
another amino acid (e.g., serine) as compared to the parent amino
acid sequence. Cysteine variants may be useful when antibodies must
be refolded into a biologically active conformation such as after
the isolation of insoluble inclusion bodies. Cysteine variants
generally have fewer cysteine residues than the native protein, and
typically have an even number to minimize interactions resulting
from unpaired cysteines.
[0035] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. In certain embodiments,
amino acid substitutions can be used to identify important residues
of antibodies to sclerostin, or to increase or decrease the
affinity of the antibodies to sclerostin described herein.
[0036] According to certain embodiments, preferred amino acid
substitutions are those which: (1) reduce susceptibility to
proteolysis, (2) reduce susceptibility to oxidation, (3) alter
binding affinity for forming protein complexes, (4) alter binding
affinities, and/or (5) confer or modify other physiochemical or
functional properties on such polypeptides. According to certain
embodiments, single or multiple amino acid substitutions (in
certain embodiments, conservative amino acid substitutions) may be
made in the naturally-occurring sequence (in certain embodiments,
in the portion of the polypeptide outside the domain(s) forming
intermolecular contacts). In certain embodiments, a conservative
amino acid substitution typically may not substantially change the
structural characteristics of the parent sequence (e.g., a
replacement amino acid should not tend to break a helix that occurs
in the parent sequence, or disrupt other types of secondary
structure that characterizes the parent sequence). Examples of
art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles
(Creighton, Ed., W.H. Freeman and Company, New York (1984));
Introduction to Protein Structure (C. Branden and J. Tooze, eds.,
Garland Publishing, New York, N.Y. (1991)); and Thornton et al.,
Nature, 354:105 (1991), which are each incorporated herein by
reference.
[0037] In certain embodiments, binding agents of the invention may
be chemically bonded with polymers, lipids, or other moieties.
[0038] The binding agents may comprise at least one of the CDRs
described herein incorporated into a biocompatible framework
structure. In one example, the biocompatible framework structure
comprises a polypeptide or portion thereof that is sufficient to
form a conformationally stable structural support, or framework, or
scaffold, which is able to display one or more sequences of amino
acids that bind to an antigen (e.g., CDRs, a variable region, etc.)
in a localized surface region. Such structures can be a naturally
occurring polypeptide or polypeptide "fold" (a structural motif),
or can have one or more modifications, such as additions, deletions
or substitutions of amino acids, relative to a naturally occurring
polypeptide or fold. These scaffolds can be derived from a
polypeptide of any species (or of more than one species), such as a
human, other mammal, other vertebrate, invertebrate, plant,
bacteria, or virus.
[0039] Typically the biocompatible framework structures are based
on protein scaffolds or skeletons other than immunoglobulin
domains. For example, those based on fibronectin, ankyrin,
lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1,
coiled coil, LAC1-D1, Z domain and tendramisat domains may be used
(see, e.g., Nygren and Uhlen, Current Opinion in Structural
Biology, 7:463-469 (1997)). In preferred embodiments, it will be
appreciated that the binding agents of the invention include the
humanized antibodies described herein. Humanized antibodies such as
those described herein can be produced using techniques known to
those skilled in the art (Zhang, W., et al., Molecular Immunology,
42(12):1445-1451 (2005); Hwang W. et al., Methods, 36(1):35-42
(2005); Dall'Acqua W F, et al., Methods, 36(1):43-60 (2005); and
Clark, M., Immunology Today, 21(8):397-402 (2000)).
[0040] The present invention therefore relates to an isolated
antibody, exemplified by antibody A but also applicable to all
antibodies disclosed herein, or an antigen binding fragment
thereof, which specifically binds to sclerostin and wherein the
variable domain of the heavy chain comprises at least one CDR
having the sequences given in SEQ ID NO:109, 115, 121, 127, 133,
139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211,
217, 223, 229, 235, 241, 247, and 253 for CDR-H1; SEQ ID NO:110,
116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188,
194, 200, 206, 212, 218, 224, 230, 236, 242, 248, and 254 for
CDR-H2; and SEQ ID NO:111, 117, 123, 129, 135, 141, 147, 153, 159,
165, 171, 177, 183, 189, 195, 201, 217, 213, 219, 225, 231, 237,
243, 249, and 255 for CDR-H3. The antibody or antigen binding
fragment thereof may comprise a heavy chain variable domain in
which the CDRs consist of at least one of the peptides of SEQ ID
NO:109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181,
187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, and 253 for
CDR-H1; SEQ ID NO:110, 116, 122, 128, 134, 140, 146, 152, 158, 164,
170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242,
248, and 254 for CDR-H2; and SEQ ID NO:111, 117, 123, 129, 135,
141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 217, 213,
219, 225, 231, 237, 243, 249, and 255 for CDR-H3.
[0041] When a light chain is present in antibodies of the
invention, the light chain may be any suitable complementary chain
and may in particular be selected from a light chain wherein the
variable domain comprises at least one or two or all of the CDRs
consisting of (or comprising) at least one of the peptides of SEQ
ID NO:106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172,
178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, and 250
for CDR-L1; SEQ ID NO:107, 113, 119, 125, 131, 137, 143, 149, 155,
161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233,
239, 245, and 251 for CDR-L2 and SEQ ID NO:108, 114, 120, 126, 132,
138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210,
216, 222, 228, 234, 240, 246, and 252 for CDR-L3.
[0042] Where an antibody comprises one or more of CDR-H1, CDR-H2,
CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as described above, it may be
obtained by expression from a host cell containing DNA coding for
these sequences. A DNA coding for each CDR sequence may be
determined on the basis of the amino acid sequence of the CDR and
synthesized together with any desired antibody variable region
framework and constant region DNA sequences using oligonucleotide
synthesis techniques, site-directed mutagenesis and polymerase
chain reaction (PCR) techniques as appropriate. DNA coding for
variable region frameworks and constant regions is widely available
to those skilled in the art from genetic sequences databases such
as GenBank.RTM.. Each of the above-mentioned CDRs will be typically
located in a variable region framework at positions 31-35 (CDR-H1),
50-65 (CDR-H2) and 95-102 (CDR-H3) of the heavy chain and positions
24-34 (CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3) of the light
chain according to the Kabat numbering system (Kabat et al., 1987
in Sequences of Proteins of Immunological Interest, U.S. Department
of Health and Human Services, NIH, USA).
[0043] Once synthesized, the DNA encoding an antibody of the
invention or fragment thereof may be propagated and expressed
according to any of a variety of well-known procedures for nucleic
acid excision, ligation, transformation, and transfection using any
number of known expression vectors. Thus, in certain embodiments,
expression of an antibody fragment may be preferred in a
prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et
al., Methods Enzymol., 178:497-515 (1989)). In certain other
embodiments, expression of the antibody or a fragment thereof may
be preferred in a eukaryotic host cell, including yeast (e.g.,
Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia
pastoris), animal cells (including mammalian cells) or plant cells.
Examples of suitable animal cells include, but are not limited to,
myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells.
Examples of plant cells include tobacco, corn, soybean, and rice
cells.
[0044] One or more replicable expression vectors containing DNA
encoding an antibody variable and/or constant region may be
prepared and used to transform an appropriate cell line, for
example, a non-producing myeloma cell line, such as a mouse NSO
line or a bacteria, such as E. coli, in which production of the
antibody will occur. In order to obtain efficient transcription and
translation, the DNA sequence in each vector should include
appropriate regulatory sequences, particularly a promoter and
leader sequence operatively linked to the variable domain sequence.
Particular methods for producing antibodies in this way are
generally well-known and routinely used. For example, basic
molecular biology procedures are described by Maniatis et al.,
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory, New York (1989). See also Maniatis et al, 3d ed., Cold
Spring Harbor Laboratory, New York (2001). DNA sequencing can be
performed as described in Sanger et al., PNAS, 74:5463 (1977), and
the Amersham International plc sequencing handbook, and site
directed mutagenesis can be carried out according to methods known
in the art (Kramer et al., Nucleic Acids Res., 12:9441 (1984);
Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-92 (1985); Kunkel et
al., Methods in Enzymol., 154:367-82 (1987); the Anglian
Biotechnology Ltd handbook). Additionally, numerous publications
describe techniques suitable for the preparation of antibodies by
manipulation of DNA, creation of expression vectors, and
transformation and culture of appropriate cells (Mountain, A. and
Adair, J. R., in Biotechnology and Genetic Engineering Reviews,
Tombs, M. P. (ed.), 10, Chapter 1, Intercept, Andover, UK (1992));
Current Protocols in Molecular Biology, F. M. Ausubel (ed.), Wiley
Interscience, New York (1999)).
[0045] Antibodies with improved affinities containing one or more
of the above-mentioned CDRs can be obtained by a number of affinity
maturation protocols including maintaining the CDRs (Yang et al.,
J. Mol. Biol., 254:392-403 (1995)), chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), use of mutation strains of E.
coli. (Low et al., J. Mol. Biol., 250:350-368 (1996)), DNA
shuffling (Patten et al., Curr. Opin. Biotechnol., 8:724-733
(1997)), phage display (Thompson et al., J. Mol. Biol., 256:7-88
(1996)) and sexual PCR (Crameri et al., Nature, 391:288-291
(1998)). All of these methods of affinity maturation are discussed
by Vaughan et al. (Nature Biotechnology, 16:535-539 (1998)).
[0046] Other antibodies according to the invention may be obtained
by conventional immunization and cell fusion procedures as
described herein and known in the art.
[0047] Monoclonal antibodies of the invention may be generated
using a variety of known techniques. In general, monoclonal
antibodies that bind to specific antigens may be obtained by
methods known to those skilled in the art (see, for example, Kohler
et al., Nature 256:495 (1975); Coligan et al. (eds.), Current
Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991);
U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993;
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.)
(1980); and Antibodies: A Laboratory Manual, Harlow and Lane
(eds.), Cold Spring Harbor Laboratory Press (1988); Picksley et
al., "Production of monoclonal antibodies against proteins
expressed in E. coli," in DNA Cloning 2: Expression Systems, 2d
Ed., Glover et al. (eds.), page 93 (Oxford University Press 1995)).
Antibody fragments may be derived therefrom using any suitable
standard technique such as proteolytic digestion, or optionally, by
proteolytic digestion (for example, using papain or pepsin)
followed by mild reduction of disulfide bonds and alkylation.
Alternatively, such fragments may also be generated by recombinant
genetic engineering techniques as described herein.
[0048] Monoclonal antibodies can be obtained by injecting an
animal, for example, a rat, hamster, a rabbit, or preferably a
mouse, including for example a transgenic or a knock-out, as known
in the art, with an immunogen comprising human sclerostin of SEQ ID
NO:1, or a fragment thereof, according to methods known in the art
and described herein. Specific antibody production may be monitored
after the initial injection and/or after a booster injection by
obtaining a serum sample and detecting the presence of an antibody
that binds to human sclerostin (or fragment thereof) using any one
of several immunodetection methods known in the art and described
herein. From animals producing the desired antibodies, lymphoid
cells, most commonly cells from the spleen or lymph node, are
removed to obtain B-lymphocytes. The B-lymphocytes are then fused
with a drug-sensitized myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal and that optionally has
other desirable properties (e.g., inability to express endogenous
Ig gene products, e.g., P3.times.63-Ag 8.653 (ATCC No. CRL 1580);
NSO, SP20) to produce hybridomas, which are immortal eukaryotic
cell lines. The lymphoid (e.g., spleen) cells and the myeloma cells
may be combined for a few minutes with a membrane fusion-promoting
agent, such as polyethylene glycol or a nonionic detergent, and
then plated at low density on a selective medium that supports the
growth of hybridoma cells but not unfused myeloma cells. A
preferred selection media is HAT (hypoxanthine, aminopterin,
thymidine). After a sufficient time, usually about one to two
weeks, colonies of cells are observed. Single colonies are
isolated, and antibodies produced by the cells may be tested for
binding activity to human sclerostin, using any one of a variety of
immunoassays known in the art and described herein. The hybridomas
are cloned (e.g., by limited dilution cloning or by soft agar
plaque isolation) and positive clones that produce an antibody
specific to sclerostin are selected and cultured. The monoclonal
antibodies from the hybridoma cultures may be isolated from the
supernatants of hybridoma cultures. An alternative method for
production of a murine monoclonal antibody is to inject the
hybridoma cells into the peritoneal cavity of a syngeneic mouse,
for example, a mouse that has been treated (e.g., pristane-primed)
to promote formation of ascites fluid containing the monoclonal
antibody. Monoclonal antibodies can be isolated and purified by a
variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography
(see, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G
(IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104
(Humana Press, Inc. (1992)). Monoclonal antibodies may be purified
by affinity chromatography using an appropriate ligand selected
based on particular properties of the antibody (e.g., heavy or
light chain isotype, binding specificity, etc.). Examples of a
suitable ligand, immobilized on a solid support, include Protein A,
Protein G, an anticonstant region (light chain or heavy chain)
antibody, an anti-idiotype antibody, and a TGF-beta binding
protein, or fragment or variant thereof.
[0049] An antibody of the present invention may also be a human
monoclonal antibody. Human monoclonal antibodies may be generated
by any number of techniques with which those having ordinary skill
in the art will be familiar. Such methods include, but are not
limited to, Epstein Barr Virus (EBV) transformation of human
peripheral blood cells (e.g., containing B lymphocytes), in vitro
immunization of human B cells, fusion of spleen cells from
immunized transgenic mice carrying inserted human immunoglobulin
genes, isolation from human immunoglobulin V region phage
libraries, or other procedures as known in the art and based on the
disclosure herein. For example, human monoclonal antibodies may be
obtained from transgenic mice that have been engineered to produce
specific human antibodies in response to antigenic challenge.
Methods for obtaining human antibodies from transgenic mice are
described, for example, by Green et al., Nature Genet., 7:13
(1994); Lonberg et al., Nature, 368:856 (1994); Taylor et al., Int.
Immun., 6:579 (1994); U.S. Pat. No. 5,877,397; Bruggemann et al.,
Curr. Opin. Biotechnol., 8:455-58 (1997); Jakobovits et al., Ann.
N.Y. Acad. Sci., 764:525-35 (1995). In this technique, elements of
the human heavy and light chain locus are introduced into strains
of mice derived from embryonic stem cell lines that contain
targeted disruptions of the endogenous heavy chain and light chain
loci (see also Bruggemann et A, Curr. Opin. Biotechnol., 8:455-58
(1997)). For example, human immunoglobulin transgenes may be
mini-gene constructs, or transloci on yeast artificial chromosomes,
which undergo B cell-specific DNA rearrangement and hypermutation
in the mouse lymphoid tissue. Human monoclonal antibodies may be
obtained by immunizing the transgenic mice, which may then produce
human antibodies specific for sclerostin. Lymphoid cells of the
immunized transgenic mice can be used to produce human
antibody-secreting hybridomas according to the methods described
herein. Polyclonal sera containing human antibodies may also be
obtained from the blood of the immunized animals.
[0050] Another method for generating human antibodies of the
invention includes immortalizing human peripheral blood cells by
EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such an
immortalized B cell line (or lymphoblastoid cell line) producing a
monoclonal antibody that specifically binds to sclerostin can be
identified by immunodetection methods as provided herein, for
example, an ELISA, and then isolated by standard cloning
techniques. The stability of the lymphoblastoid cell line producing
an anti-sclerostin antibody may be improved by fusing the
transformed cell line with a murine myeloma to produce a
mouse-human hybrid cell line according to methods known in the art
(see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)). Still
another method to generate human monoclonal antibodies is in vitro
immunization, which includes priming human splenic B cells with
human sclerostin, followed by fusion of primed B cells with a
heterohybrid fusion partner. See, e.g., Boerner et al., J.
Immunol., 147:86-95 (1991).
[0051] In certain embodiments, a B cell that is producing an
anti-human sclerostin antibody is selected and the light chain and
heavy chain variable regions are cloned from the B cell according
to molecular biology techniques known in the art (WO 92/02551; U.S.
Pat. No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA,
93:7843-48 (1996)) and described herein. B cells from an immunized
animal may be isolated from the spleen, lymph node, or peripheral
blood sample by selecting a cell that is producing an antibody that
specifically binds to sclerostin. B cells may also be isolated from
humans, for example, from a peripheral blood sample. Methods for
detecting single B cells that are producing an antibody with the
desired specificity are well known in the art, for example, by
plaque formation, fluorescence-activated cell sorting, in vitro
stimulation followed by detection of specific antibody, and the
like. Methods for selecting specific antibody-producing B cells
include, for example, preparing a single cell suspension of B cells
in soft agar that contains human sclerostin. Binding of the
specific antibody produced by the B cell to the antigen results in
the formation of a complex, which may be visible as an
immunoprecipitate. After the B cells producing the desired antibody
are selected, the specific antibody genes may be cloned by
isolating and amplifying DNA or mRNA according to methods known in
the art and described herein.
[0052] An additional method for obtaining antibodies of the
invention is by phage display. See, e.g., Winter et al., Annu. Rev.
Immunol., 12:433-55 (1994); Burton et al., Adv. Immunol.,
57:191-280 (1994). Human or murine immunoglobulin variable region
gene combinatorial libraries may be created in phage vectors that
can be screened to select Ig fragments (Fab, Fv, sFv, or multimers
thereof) that bind specifically to TGF-beta binding protein or
variant or fragment thereof. See, e.g., U.S. Pat. No. 5,223,409;
Huse et al., Science, 246:1275-81 (1989); Sastry et al., Proc.
Natl. Acad. Sci. USA, 86:5728-32 (1989); Alting-Mees et al.,
Strategies in Molecular Biology, 3:1-9 (1990); Kang et al., Proc.
Natl. Acad. Sci. USA, 88:4363-66 (1991); Hoogenboom et al., J.
Molec. Biol., 227:381-388 (1992); Schlebusch et al., Hybridoma,
16:47-52 (1997) and references cited therein. For example, a
library containing a plurality of polynucleotide sequences encoding
Ig variable region fragments may be inserted into the genome of a
filamentous bacteriophage, such as M13 or a variant thereof, in
frame with the sequence encoding a phage coat protein. A fusion
protein may be a fusion of the coat protein with the light chain
variable region domain and/or with the heavy chain variable region
domain. According to certain embodiments, immunoglobulin Fab
fragments may also be displayed on a phage particle (see, e.g.,
U.S. Pat. No. 5,698,426).
[0053] Heavy and light chain immunoglobulin cDNA expression
libraries may also be prepared in lambda phage, for example, using
.lamda.lmmunoZap.TM.(H) and .lamda.ImmunoZap.TM.(L) vectors
(Stratagene, La Jolla, Calif.). Briefly, mRNA is isolated from a B
cell population, and used to create heavy and light chain
immunoglobulin cDNA expression libraries in the .lamda.ImmunoZap(H)
and .lamda.ImmunoZap(L) vectors. These vectors may be screened
individually or co-expressed to form Fab fragments or antibodies
(see Huse et al., supra; see also Sastry et al., supra). Positive
plaques may subsequently be converted to a non-lytic plasmid that
allows high level expression of monoclonal antibody fragments from
E. coli.
[0054] In one embodiment, in a hybridoma the variable regions of a
gene expressing a monoclonal antibody of interest are amplified
using nucleotide primers. These primers may be synthesized by one
of ordinary skill in the art, or may be purchased from commercially
available sources. See, e.g., Stratagene (La Jolla, Calif.), which
sells primers for mouse and human variable regions including, among
others, primers for V.sub.Ha, V.sub.Hb, V.sub.Hc, V.sub.Hd,
C.sub.Hl, V.sub.L and C.sub.L regions. These primers may be used to
amplify heavy or light chain variable regions, which may then be
inserted into vectors such as ImmunoZAP.TM.H or ImmunoZAP.TM.L
(Stratagene), respectively. These vectors may then be introduced
into E. coli, yeast, or mammalian-based systems for expression.
Large amounts of a single-chain protein containing a fusion of the
V.sub.H and V.sub.L domains may be produced using these methods
(see Bird et al., Science, 242:423-426, (1988)).
[0055] Once cells producing antibodies according to the invention
have been obtained using any of the above-described immunization
and other techniques, the specific antibody genes may be cloned by
isolating and amplifying DNA or mRNA therefrom according to
standard procedures as described herein. The antibodies produced
therefrom may be sequenced and the CDRs identified and the DNA
coding for the CDRs may be manipulated as described previously to
generate other antibodies according to the invention.
[0056] Preferably the binding agents specifically bind to
sclerostin. As with all binding agents and binding assays, one of
skill in this art recognizes that the various moieties to which a
binding agent should not detectably bind in order to be
therapeutically effective and suitable would be exhaustive and
impractical to list. Therefore, for a binding agent disclosed
herein, the term "specifically binds" refers to the ability of a
binding agent to bind to sclerostin, preferably human sclerostin,
with greater affinity than it binds to an unrelated control
protein. Preferably the control protein is hen egg white lysozyme.
Preferably the binding agents bind to sclerostin with an affinity
that is at least, 50, 100, 250, 500, 1000, or 10,000 times greater
than the affinity for a control protein. A binding agent may have a
binding affinity for human sclerostin of less than or equal to
1.times.10.sup.-7M, less than or equal to 1.times.10.sup.-8M, less
than or equal to 1.times.10.sup.-9M, less than or equal to
1.times.10.sup.-10 M, less than or equal to 1.times.10.sup.-11M, or
less than or equal to 1.times.10.sup.-12M.
[0057] Affinity may be determined by an affinity ELISA assay. In
certain embodiments, affinity may be determined by a BIAcore assay.
In certain embodiments, affinity may be determined by a kinetic
method. In certain embodiments, affinity may be determined by an
equilibrium/solution method. Such methods are described in further
detail herein or known in the art.
[0058] Sclerostin binding agents of the present invention
preferably modulate sclerostin function in the cell-based assay
described herein and/or the in vivo assay described herein and/or
bind to one or more of the epitopes described herein and/or
cross-block the binding of one of the antibodies described in this
application and/or are cross-blocked from binding sclerostin by one
of the antibodies described in this application. Accordingly such
binding agents can be identified using the assays described
herein.
[0059] In certain embodiments, binding agents are generated by
first identifying antibodies that bind to one more of the epitopes
provided herein and/or neutralize in the cell-based and/or in vivo
assays described herein and/or cross-block the antibodies described
in this application and/or are cross-blocked from binding
sclerostin by one of the antibodies described in this application.
The CDR regions from these antibodies are then used to insert into
appropriate biocompatible frameworks to generate sclerostin binding
agents. The non-CDR portion of the binding agent may be composed of
amino acids, or may be a non-protein molecule. The assays described
herein allow the characterization of binding agents. Preferably the
binding agents of the present invention are antibodies as defined
herein.
[0060] It will be understood by one skilled in the art that some
proteins, such as antibodies, may undergo a variety of
posttranslational modifications. The type and extent of these
modifications often depends on the host cell line used to express
the protein as well as the culture conditions. Such modifications
may include variations in glycosylation, methionine oxidation,
diketopiperizine formation, aspartate isomerization and asparagine
deamidation. A frequent modification is the loss of a
carboxy-terminal basic residue (such as lysine or arginine) due to
the action of carboxypeptidases (as described in Harris, R. J.,
Journal of Chromatography, 705:129-134 (1995).
[0061] To generate antibodies A-M, rabbits were immunized by
subcutaneous injection of recombinant human sclerostin at three
weekly intervals, initially with Freund's Complete Adjuvant and
subsequently with Freund's Incomplete Adjuvant. Peripheral blood
lymphocytes were harvested and purified on Lympholyte-R (Cedarlane
Labs). Immune rabbit lymphocytes were cultures in the presence of
irradiated EL-4 cells and rabbit T cell conditioned media, before
supernatants were screened for binding to human sclerostin.
Individual B cells secreting antibody with appropriate binding
characteristics were isolated from positive microtiter wells
according to the Selected Lymphocyte Antibody Method (Babcook et
al., Proc. Natl. Acad. Sci. USA, 93:784307848 (1996)), and heavy
and light chain variable region genes were cloned from single
rabbit B cells. Variable regions were expressed as recombinant
chimeric IgGs to confirm binding.
[0062] In the amino acid sequences shown below, the signal peptides
are underlined and the boxed-shaded amino acids represent
complement-determining regions (CDR's).
TABLE-US-00001 Antibody A VL ##STR00001## (SEQ ID NO: 3)
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCAGCCGTGCTGACCCAGACACCATCGCCCGTGT
CTGCAGGTGTGGGAGGCACAGTCACCATCAAGTGCCAGTCCAGTCAGAGG
GTTTTTAATAACAACGAATTATCCTGGTATCAGCAGAAACCAGGGCAGCC
GCCCAAACGCCTGATCTATGAAGCATCCACACTGGCATCTGGGGTCCCAG
ATAGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGC
GGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGA
TGATGATGGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC GT VH
##STR00002## (SEQ ID NO: 5)
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTG
GGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAATAAT
TACTATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATGGAT
CGGACTCATTTATCCTGATGGTAGCACATTCTACGCGAACTGGGCGGAAG
GCCGATTCACCAGCTCCAAGACCTCGACCACGGTGACTCTGAAGATGACC
AGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGAAGGTGG
TGCTGGTGATAATACTGGTACCGAATATTACTACGGCGTGGACCTCTGGG
GCCAGGGCACCCTCGTCACCGTCTCGAGC Antibody B VL ##STR00003## (SEQ ID
NO: 7) ATGGACATGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCCATCGTGATGACCCAGACTCCATCTTCCGTGT
CTGCAGCTGTGGGAGATACAGTCACCATCAATTGCCAGGCCAGTGAGAAC
GTTTATGATAAAAGCGCCTTATCCTGGTATCAGCAGAAACCAGGGCAGCC
TCCCAAGCTCCTGATCTATCTGGCATCCACTCTGGCATCTGGGGTCCCAT
CGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGC
GATGTGGTGTGTGCGGATGCTGCCACTTACTACTGTGCAGGATATAAAAG
TAGTATTATTGATGGTACTGCTTTCGGCGGAGGGACCGACGTGGTGGTCA AAGGA VH
##STR00004## (SEQ ID NO: 9)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGC
CTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGT
AACAATGCACTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATG
GATCGGGGCCATTGGTGCTGGTGGTAACACATACTATGCGAGCTGGGCGA
AAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAGA
ATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAACGG
GGACCTGCCAGGTGGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCC TA Antibody C VL
##STR00005## (SEQ ID NO: 11)
ATGGACATAAGGGCCCCCACTCAGCTGCTGGGGCTCCTACTGCTCTGGCT
CCCAGGTGCCAGATGTGCTGACATTGTGATGACCCAGACTGCATCCCCCG
TGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAG
AGCATTAGCCCTGCATTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCC
CAAGCTCCTGATCTACGATGTATCGAAACTGGCATCTGGGGTCCCATCGC
GGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGAC
CTGGAGTGTGCCGATGGTGCCACTTACTACTGTCAAAGCTATTATGGTAT
TAATAGTAATAGTTATGGTAATATTTTCGGCGGAGGGACCGAGGTGGTGG TCAAAGGA VH
##STR00006## (SEQ ID NO: 13)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTAT
GCAATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGG
CTACATTTATGGTAATTATAATAAATACTACGCGAGCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCTCGACCATGGTGGATCTGAAGATGACTAGT
CTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGGGGTGCTAT
GGATGTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCAGC Antibody D VL ##STR00007##
(SEQ ID NO: 15) ATGGACACCAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGCGCCTATGATATGACCCAGACTCCAGCCTCTGTGG
AGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAGC
ATTAGCACTTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAA
GCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGT
TCAAAGGCAGTGGATATGGGACACAGTTCACTCTCACCATCAGCGGCGTG
GAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTGGAATATTAA
TAATATTGATAATATTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGA VH ##STR00008##
(SEQ ID NO: 17) ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTAT
TCAATGTATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGG
ATTTATTCTTAGTGCTACTGCCGTATCCTACGCGACCTGGGCGAAAGGCC
GATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGT
CCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATAGAGATGG
TGGTACTACACTAGATGGTTTTGATCCCTGGGGCCCAGGCACCCTGGTCA CCGTCTCCAGC
Antibody E VL ##STR00009## (SEQ ID NO: 19)
ATGGACATCAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCCCAACTGCTGACCCAGACTCCACCCTCGGTGT
CTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGT
GTTGTTAATAACAACAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCC
TCCCAAGCTCCTGATCTATTTTGCCTCCACTCTGGCATCTGGGGTCCCAT
CGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGC
GACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCACTTATCT
TAGTGATGATTGGTCCGATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA AAGGA VH
##STR00010## (SEQ ID NO: 21)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCAAAGTCTCTGGATTCTCCCTCAGTTCCTCT
GTAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGG
AGCCATTTGGAGTGGTGGTTACACATACTACGCGACCTGGGCAAAAGGCC
GATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATGACCAGT
CTGACAGCCGCGGACACGGCCACCTACTTCTGTGCCAGAGGACAATTTGG
TGCTAGTGGTGGTGGTGATGTTTTGTGGGGCCCGGGCACCCTGGTCACCG TCTCCAGC
Antibody F VL ##STR00011## (SEQ ID NO: 23)
ATGGACATAAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCTCCGTGT
TCTGCAGCTGGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCCGAGT
GTTGATAATAACAACCTCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCC
TCCCAAGCTCCTGATCTATGATGCTTCCACTCTGGCATCTGGGGTCCCAT
CGCGGTTCGAAGGCAGTGGATCTGGGACACAATTCACTCTCGCCATCAGC
GGCGTTCAGTGTGACGATGCTGCCACTTACTACTGTGTAGCCGGTTATGG
TAAAAGGAGTCGAGATATACGAGTTTTCGGCGGAGGGACCGGGGTGGTGG TCAAAGGA VH
##STR00012## (SEQ ID NO: 25)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACCGTCTCTGGATTCGACATCAGTAGCCAC
AATATGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGG
AATCATTTATCCTAGTAATAATGCATACTATTCCAACTGGGCGAAAGGCC
GATTCACCATCTCCAAAACTTCGACCACGATGTCTCTGCAAATGAACAGT
CTGACAACCGAGGACACGGCCACCTATTTCTGCGCCAGAGACATAAATAG
TGCTATTTGGGGCCCAGGCACCCTGGTCACCGTCTCCAGC Antibody G VL ##STR00013##
(SEQ ID NO: 27) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGTCATATGTGCCGAAGTAGCGATGACCCAGACTCCATCCTCCG
TGTCTGCAGCTGTGGGAGGCACAGTCACCATCGATTGCCAGGCCAGTGAG
AGCATTTATAGCAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCC
CAAGCTCCTGATCTATGATGCATCCGATCTGGCATCTGGGGTCCCATCGC
GGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGAC
CTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTAGTTGGGGTGG
TAGTACTTATGGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC GT VH
##STR00014## (SEQ ID NO: 29)
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATCTAT
GTAATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGG
AAGCATCGATGCTGACGATAGCGCATACTACGCGACCTGGGCGACAAGCC
GATCCACCATCTCCAGAACCTCGACCACGGTGGCTCTGAGCATCACCAGT
CCGACAACCGAGGACACGGCGACCTATTTCTGTGCCAGGGGACTATATGC
TAATGGTGGTCCCTTTACCTTATGGGGCCCAGGCACCCTCGTCACCGTCT CGAGC Antibody H
VL ##STR00015## (SEQ ID NO: 31)
ATGGACATGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCCGCCGTGCTGACCCAGACTCCATCTCCCGTGT
CTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGTCCAGTCCGAGT
GTTTATAATCGCAACCAATTATCCTGGTTTCAGCAGAAAGCAGGGCAGCC
TCCCAAGCTCCTGATCTTTACTGCGTCCACTCTGGCATCTGGGGTCCCAT
CGCGGTTCAAAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
GACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAGGCTATTATAA
TACTGGTAGTGATACGTATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA AAGGA VH
##STR00016## (SEQ ID NO: 33)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAATAACTAC
GACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATATATCGG
ATTCATTAATACTGTTGGTTACGCATACTACGCGAGCTGGGCAAAAGGCC
GATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATGACCAGT
CTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGCCTTGATAATTA
CTATACTTGGGGCATCTGGGGCCCAGGCACCCTGGTCACCGTTTCCCTA Antibody I VL
##STR00017## (SEQ ID NO: 35)
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGTGCCGACATCGTGATGACCCAGACTCCAGGCTCCG
TGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCGCTGAG
GACATTTATAGCTCTTTGGCCTGGTATCAGCAGAAATCAGGGCAGCCTCC
CAAGCTCCTGATCTATGCTGCATCTATTCTGGCATCTGGGGCCCCATCGC
GGTTCAGTGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGGC
GTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAACCAATTATGGTAT
CAGTAGTTATGGTGCGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAG GA VH
##STR00018## (SEQ ID NO: 37)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACCGTCTCTGGGTTCTCCCTCAGTAACAAG
CCAATAACATGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAATACATCGG
ATGGATTAGTACTACTGGTAGCGCATACTACGCGAGCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGT
CCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATATAGTAGTGA
TTATGGACATCATGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCA GC Antibody J VL
##STR00019## (SEQ ID NO: 39)
ATGGACACCAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCACCCGTGT
CTGCAGCTCTGGGAGGCTCAGTCACCATCAATTGCCAGGCCAGTCAGAGT
GTTTATAGAGATTACTTATCCTGGTTTCAGCAGAAACCAGGTCAGCCTCC
CAAGCTCCTGATCAATGGTGCATCCAATCTGGCATCTGGGGTCCCATCGC
GGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGAC
GTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTTTAGTGG
TAATATCAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAGGGA VH ##STR00020## (SEQ
ID NO: 41) ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAG
GATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAATAACTAC
CACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGAAATACATCGG
AATCATTAGTAATACTGGTTACACATACTACTCGAGCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACC
AGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCTGGAGACCGGCT
TGCTAACTTGTGGGGCCCAGGCACCCTGGTCACTGTCTCCAGC Antibody K VL
##STR00021## (SEQ ID NO: 43)
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGTGCCGACGTCGTGATGACCCAGACTCCAGCCTCCG
TGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAG
AGCATTAGTACCTACTTAAAGTGGTATCAGCAGAAACCAGGACAGCGTCC
CAAGCGCCTGATCTATTCTGCATCCACTCTGACATCTGGGGTCCCATCGC
GGTTCAAAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGAC
CTGGAGTGTGGCGATGCTGCCACTTACTACTGTCAAAGCAATGCTGGTAG
TAGTAGTAGTAGTTGTGGTTATGCTTTCGGCGGAGGGACCGAGGTGGTGG TCAAAGGA VH
##STR00022## (SEQ ID NO: 45)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGAATTGACCTCAGTTACTAT
GGGATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGG
GATCATTAGTGGTATTGGTAATACATACTATCCGACCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCCCGACCACGGTGGATCTAAGGATCACCAGT
CCGACAACCGAGGACACGGCCACCTATTTCTGTGCCACTGGGGACTTCTG
GGGCCCAGGCACCCTGGTCACCGTCTCCAGC Antibody L VL ##STR00023## (SEQ ID
NO: 47) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGTGCCGACGTCGTGATGACCCAGACTCCAGCCTCCG
TGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAG
AGCATTAGTAGGTACTTAAAGTGGTATCAGCAGAAACCAGGGCAGCGTCC
CAAGCGCCTGATCTATTCTGCATCCACTCTGACATCTGGGGTCCCATCGC
GGTTCAAAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGAC
CTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCAATGCTGGTAG
TAGCAGTAGTAGTTGTGGTTATGCTTTCGGCGGAGGGACCGAGGTGGTGG
TCAAAGGA VH ##STR00024## (SEQ ID NO: 49)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGTCTCTGGAGTCGACCTCAGTTACTAT
GGAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGG
GATCATTAGTGGTAGTGGTAACACATACTACGCGAGCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGT
CCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGTGGGGACTTCTG
GGGCCCAGGCACCCTGGTCACCGTCTCCAGC Antibody M VL ##STR00025## (SEQ ID
NO: 51) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGTGCCCTTGTGATGACCCAGACTGCATCCCCCGTGT
CTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGT
GTTTATAGTAACTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCC
CAAGCTCCTGATCTATGTTGCATCCAGTCTGGCATCTGGGGTCCCATCGC
GATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGTGAC
CTGGAGTGTGACGATGCTGCCACTTACTACTGTGGAGGCTTTCAGAAATA
TATTGATGATGGGGGGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAG GA VH
##STR00026## (SEQ ID NO: 53)
ATGGAGACAGGCCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAG
GATCCCTGACACTTACCTGCACAGTCTCTGGAATCGACCTCAGTACCTAT
GCAATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGG
AATCATGAGTAGTAGTGGTAGCGCATACTACGCGAGCTGGGCGAAAGGCC
GATTCACCATCTCCAAAACCGCGTCGACCACGGTGGATCTGAAAATGACC
AGTCTGACAATCGAGGACACGGCCACCTATTTCTGTGCCAGAAGTTCTAG
TTTTGGATTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCAGC
[0063] Antibodies N-Y were generated in mice. The Kappa Constant
region for all VK regions of antibodies N-Y is as follows:
TABLE-US-00002 (SEQ ID NO: 54)
TDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN
GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC
[0064] The Heavy Constant Region for all VH regions of antibodies
N-Y is as follows:
TABLE-US-00003 (SEQ ID NO: 55)
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVL
QSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVS
SVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQF
NSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK
EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN
VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
[0065] In the following antibody amino acid sequences, the
boxed-shaded amino acids represent complement-determining regions
(CDRs) and the underlined amino acids represent signal peptide.
TABLE-US-00004 Antibody N VK ##STR00027## (SEQ ID NO: 57)
ATGGATTTTCAAGTGCAGATTTTCAGCTTCATGCTAATCAGTGTCACAGT
CATATTGTCCAGTGGAGAAATTGTGCTCACCCAGTCTCCAGCACTCATGG
CTGCATCTCCAGGGGAGAAGGTCACCATCGCCTGCAGTGTCAGCTCAAGT
ATAAGTTCCACCAACTTACACTGGTCCCAGCAGAAGTCAGGAACCTCCCC
CAAACTCTGGATTTATGGCACATCCAACCTTGCTTCTGGAGTCCCTGTTC
GCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGC
ATGGAGGCTGAAGATGCTGCCACCTATTACTGTCAACAGTGGAGTACTAC
GTATACGTTCGGATCGGGGACCAAGCTGGAGCTGAAACGT VH ##STR00028## (SEQ ID
NO: 59) ATGGGATGGAACTGGATCATCTTCTTCCTGATGGCAGTGGTTACAGGGGT
CAATTCAGAGGTGCAGTTGCAGCAGTCTGGGGCAGACCTTGTGAAGCCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTATATACACTGGGTGAAGCAGAGGCCTGCACAGGGCCTGGAGTGGAT
TGGAAGGATTGATCCTGATAATGGTGAAAGTACATATGTCCCGAAGTTCC
AGGGCAAGGCCACTATAACAGCAGACACATCATCCAACACAGCCTACCTA
CAACTCAGAAGCCTGACATCTGAGGACACTGCCATCTATTATTGTGGGAG
AGAGGGGCTCGACTATGGTGACTACTATGCTGTGGACTACTGGGGTCAAG
GAACCTCAGTCACTGTCTCGAGC Antibody O VK ##STR00029## (SEQ ID NO: 61)
ATGGAGTCAGACACACTCCTGCTATGGGTGCTACTGCTCTGGGTTCCAGG
TTCCACAGGTAAAATTGTACTGACCCAATCTCCAGCTTCTTTGGCTGTGT
CTCTAAGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGAT
AGTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCC
ACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGGTCCCTG
CCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGAT
CCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATTATGA
GGATCCGCTCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAACGT VH ##STR00030## (SEQ
ID NO: 63) ATGGGATGGCCCTGGGTCTTTATATTTCTCCTGTCAGTAACTGCAGGTGT
TCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTG
GGACCTCAGTGAAGGTGTCCTGCAAGGCCTCTGGATACGCCTTCACTAAT
TACTTGATAGAGTGGATAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGAT
TGGAGTGATTAATCCTGGAAGCGGTATTATTAATTACAATGAGAAGTTCA
AAATCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATG
CAGCTCAGCAGCCTGACATCTGATGATTCTGCGGTCTATTTCTGTGCAAG
AGACTGGGATACTTTCTATAGTTACGAGAGGGAGGTCTATGCTATGGACT
ACTGGGGTCAAGGGACCTCAGTCACCGTCTCGAGC Antibody P VK ##STR00031## (SEQ
ID NO: 65) ATGGAATCACAGACTCTGGTCCTTGTATTTTTGCTTTTCTGGATTCCAGC
CTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGA
GTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGC
ACAAACATACACTGGTATCAGCAAAGAACAGATGGTTCTCCAAGGCTTCT
CATAAAGTTTGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTG
GCAGTGGCTCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCT
GAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCACTCAC
GTTCGGTGCTGGGACCAAGCTGGAACTGAAACGT VH ##STR00032## (SEQ ID NO: 67)
ATGGATTGGAGCTGGATCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTGAAGAC
TACTATGTGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATATGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGG
GGGGTATGGTAACTTTTACTTTGACTACTGGGGCCAAGGCACCACTGTCA CCGTCTCGAGC
Antibody Q VK ##STR00033## (SEQ ID NO: 69)
ATGGAATTCCAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGG
TGTTGAAGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACAT
CAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGT
ACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACT
GATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAG
GCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCT
GAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCCGTACAC
GTTCGGAGGGGGGACCAAGCTGGAGCTGAAACG VH ##STR00034## (SEQ ID NO: 71)
ATGAAATGCAACTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTTTATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATATGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
AGGGCAGGCAACTTACTACTTTGACTACTGGGGCCAAGGCACCACGGTCA CCGTCTCGAGC
Antibody R VK ##STR00035## (SEQ ID NO: 73)
ATGGAGACAGACACACTCCGAGCGAAGCTTCGAAGCCACCATGAAGCTGC
CTGTTCTGCTTTTAGGTGTGAGATGTGACATCCAGATGAACCAGTCTCCA
TCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGC
CACTCAGAACATTAATGTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAA
ATATTCCTACACTTTTGATCTATAAGACTTCCAACTTGCACACAGGCGTC
CCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCAT
CAGCAGCCTGCAGTCTGAAGACATTGCCACTTACTACTGTCAACAGGGTC
AAAGTTTTCCGTACACGTTCGGAGGAGGCACCAAGCTGGAGCTGAAACGT VH ##STR00036##
(SEQ ID NO: 75) ATGAAATGCAACTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTTTATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATATGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
AGGGCAGGCAACTTACTACTTTGACTACTGGGGCCAAGGCACCACGGTCA CCGTCTCGAGC
Antibody S VK ##STR00037## (SEQ ID NO: 77)
ATGGAATCACAGACCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGG
TACCTGTGGGGACATTATGATGTCACAGTCTCCATCCTCCCTAGCTGTGT
CAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTA
TATAGTAACAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGG
GCAGTCTCCTAAACTGCTGATTTCCTGGGCATCCACTAGGGAATCTGGGG
TCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACC
ATCAGCAGTGTGAAGGCTGAAGACCTGGCTGTTTATTACTGTCAGCAATA
TTATGACTATCCGTGGACGTTCGGTGGAGGCTCCAAGCTGGAGCTGAAAC GT VH
##STR00038## (SEQ ID NO: 79)
ATGGGATGGAACTGGATCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTTTATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATATGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
AGGGCAGGCAACTTACTACTTTGACTACTGGGGCCAAGGCACCACGGTCA CCGTCTCGAGC
Antibody T VK ##STR00039## (SEQ ID NO: 81)
ATGGACATGAGGACCCCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTT
TCAAGGTACCAGATGTGATATCCAGATGACACAGACTTCATCTTCCCTGT
CTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGACAAGTCAGGAT
ATTAACAATTTTTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAA
ATTCCTGATCCACCACACATCAAGATTAAAGTCAGGAGTCCCATCAAGGT
TCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCCACCTG
GAACCTGAAGATATTGCCACTTACTATTGTCAGCACTATTATAATCTTCC
GTGGACGTTCGGTGGAGGCACCAAGTTGGAGCTGAAACGT VH ##STR00040## (SEQ ID
NO: 83) ATGGGATGGAGCTGGACCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT
CCCACTTGAGATCCAGCTGCAGCAGACTGGACCTGAGCTGGTGAAGCCTG
GGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGAC
AACATCATGGTCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGAT
TGGAAACATTAATCCTTACTATGGTAGTCCTAGTTACAATCTGAAGTTCA
AGGACAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATG
CAGCTCAATAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAG
AGAGGGGGGTAATTACGGTTCTCTGGACAACTGGGGTCAAGGAACCTCGG TCACCGTCTCGAGC
Antibody U VK ##STR00041## (SEQ ID NO: 85)
ATGAAGTCACAGACCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGG
TACCTGTGGGGACATTATGATGTCACAGTCTCCATCCTCCCTAGCTGTGT
CAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTA
TATAGTAACAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGG
GCAGTCTCCTAAACTGCTGATTTCCTGGGCATCCACTAGGGAATCTGGGG
TCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACC
ATCAGCAGTGTGAAGGCTGAAGACCTGGCTGTTTATTACTGTCAGCAATA
TTATGACTATCCGTGGACGTTCGGTGGAGGCTCCAAGCTGGAAATAAAAC GT VH
##STR00042## (SEQ ID NO: 87)
ATGGGATGGAGCTGGACCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT
CCACTCTGAGATCCAGCTGCAGCAGACTGGACCTGAGCTGGTGAAGCCTG
GGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGAC
AACATCATGGTCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGAT
TGGAAACATTAATCCTTACTATGGTAGTCCTAGTTACAATCTGAAGTTCA
AGGACAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATG
CAGCTCAATAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAG
AGAGGGGGGTAATTACGGTTCTCTGGACAACTGGGGTCAAGGAACCTCGG TCACCGTCTCGAGC
Antibody V VK ##STR00043## (SEQ ID NO: 89)
ATGAGTCCTGCCCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTAC
CAGATGTGATATCCAGATGACACAGACTTCATCTTCCCTGTCTGCCTCTC
TGGGAGACAGAGTCACCATCAGTTGCAGGACAAGTCAGGATATTAACAAT
TTTTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAATTCCTGAT
CCACCACACATCAAGATTAAAGTCAGGAGTCCCATCAAGGTTCAGTGGCA
GTGGGTCTGGGACAGATTATTCTCTCACCATCAGCCACCTGGAACCTGAA
GATATCGCCACTTACTATTGTCAGCACTATTATAATCTTCCGTGGACGTT
CGGTGGAGGCACAAAGTTGGAAATAAAACGT VH ##STR00044## (SEQ ID NO: 91)
ATGGGATGGAGCTGGACCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT
CCACTCTGAGGTTCAGCTCGTTGAATCCGGAGGCGGACTCGTGAAGCCCG
GGGGCAGTCTTAAACTGTCCTGCGCCGTCAGCGGGTTTTCCCTCAGTTAC
TATTACATGTCATGGGTGCGGCAGACCCCCGAGAAAAGGTTGGAATGGAT
TGGCACCATCTCAATTGACGGAAGCACATATTACGCATCTTGGGCCGAGG
GTCGCTTCACAATTTCTAAGGACAGTACTACCGTGTATCTGCAGATGTCC
AAGCCTTAAGAGCGAAGATACAGCCATGTCTACTGTGCTAGAGGGCACAT
CAACACTGGAATGGATCCTTGGGGCCAGGGCACCCCGGTCACAGTCTCGA GC Antibody W VK
##STR00045## (SEQ ID NO: 93)
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC
CAGCAGTGATGTTTTGATGACCCAAAATCCACTCTCCCTGCCTGTCAGTC
TTGGAGATCAAGCCTCCATCTCCTGCAGATCAAGTCAGACCATTGTACAT
AGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTC
TCCAAAGCTCCTGATCTACGAAGTTTCCAACCGATTTTCTGGGGTCCCAG
ACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAAATCAGC
AGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACA
TTTTCCGCACACGTTCGGAGGGGGGACCAAGCTGGAACTGAAACGT VH ##STR00046## (SEQ
ID NO: 95) ATGGAATGGAGCGGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGTTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTATATGTATTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATGTGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
CGATAGGTACGACGAGGGGGCCGCGTCGGACTATGGTATGGACTACTGGG
GTCAAGGAAGTTCAGTCACAGTCTCGAGC Antibody X VK ##STR00047## (SEQ ID
NO: 97) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC
CAGCAGTGATGTTTTGATGACCCAAAATCCACTCTCCCTGCCTGTCAGTC
TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACAT
AGTAATGGAAACACCCATTTAGAATGGTACCTGCAGAAACCAGGCCAGTC
TCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAG
ACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACA
TGCTCCGCACACGTTCGGAGGGGGGACCAAGCTGGAACTGAAACGT VH ##STR00048## (SEQ
ID NO: 99) ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTTTGGGGCAGAGTTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTATATGTATTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGGGAATGGTGATACTGAATGTGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
CGATAGATACGACGAGGGGGCCGCGTCGGACTATGCTGTGGACTACTGGG
GTCAAGGAAGTTCGGTCACAGTCTCGAGC Antibody Y VK ##STR00049## (SEQ ID
NO: 101) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC
CAGCAGTGATGTTTTGATGACCCAAAATCCACTCTCCCTGCCTGTCAGTC
TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACAT
AGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTC
TCCAAAGCTCCTGATCTACGAAGTTTCCAACCGATTTTCTGGGGTCCCAG
ACAGGTTCAGTGGCAGTGGCTCAGGGACAGATTTCACACTCAAAATCAGC
TAGAGTGGGGGCTGAAGATCTGGGAATTTATACTGCTTTCAAGGTTCACT
TTTTCCGCACACGTTCGGAGGGGGGACCAAGTTGGAGCTCAAACGT VH ##STR00050##
(SEQ ID NO: 103) ATGGAATGGAGCTGTATCATCTTCTTCCTGATGGCAGTGGTTATAGGAAT
CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGTTTGTGAGGTCAG
GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC
TACTATATGTATTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGAT
TGGATGGATTGATCCTGAGAATGGTGATACTGAATGTGCCCCGAAGTTCC
AGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGC
CGATAGATACGACGAGGGGGCCGCGTCGGACTATGCTGTGGACTACTGGG
GTCAAGGAAGCCAGTCACCGTCTCGAGC
Table 1 shows the CDR's and sequence identifiers for Antibodies
A-Y.
TABLE-US-00005 TABLE 1 Antibody CDR 1 CDR 2 CDR 3 A VL
QSSQRVFNNNELS EASTLAS LGGYDDDGDNA (SEQ ID NO: 106) (SEQ ID NO: 107)
(SEQ ID NO: 108) A VH NYYMT LIYPDGSTFYANWAEG EGGAGDNTGTEYYYGVDL
(SEQ ID NO: 109) (SEQ ID NO: 110) (SEQ ID NO: 111) B VL
QASENVYDKSALS LASTLAS AGYKSSIIDGTA (SEQ ID NO: 112) (SEQ ID NO:
113) (SEQ ID NO: 114) B VH NNALS AIGAGGNTYYASWAKG GDLPGGI (SEQ ID
NO: 115) (SEQ ID NO: 116) (SEQ ID NO: 117) C VL QASQSISPALA DVSKLAS
QSYYGINSNSYGNI (SEQ ID NO: 118) (SEQ ID NO: 119) (SEQ ID NO: 120) C
VH SYAMG YIYGNYNKYYASWAKG GGAMDV (SEQ ID NO: 121) (SEQ ID NO: 122)
(SEQ ID NO: 123) D VL QASESISTWLA RASTLAS QQGWNINNIDNI (SEQ ID NO:
124) (SEQ ID NO: 125) (SEQ ID NO: 126) D VH SYSMY FILSATAVSYATWAKG
DRDGGTTLDGFDP (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 129) E
VL QSSQSVVNNNNLA FASTLAS QGTYLSDDWSDA (SEQ ID NO: 130) (SEQ ID NO:
131) (SEQ ID NO: 132) E VH SSVMN AIWSGGYTYYATWAKG GQFGASGGGDVL (SEQ
ID NO: 133) (SEQ ID NO: 134) (SEQ ID NO: 135) F VL QSSPSVDNNNLLS
DASTLAS VAGYGKRSRDIRV (SEQ ID NO: 136) (SEQ ID NO: 137) (SEQ ID NO:
138) F VH SHNMQ IIYPSNNAYYSNWAKG DINSAI (SEQ ID NO: 139) (SEQ ID
NO: 140) (SEQ ID NO: 141) G VL QASESIYSNLA DASDLAS QCSWGGSTYGFA
(SEQ ID NO: 142) (SEQ ID NO: 143) (SEQ ID NO: 144) G VH IYVMT
SIDADDSAYYATWA GLYANGGPFTL (SEQ ID NO: 145) TS (SEQ ID NO: 147)
(SEQ ID NO: 146) H VL QSSPSVYNRNQLS TASTLAS QGYYNTGSDTYA (SEQ ID
NO: 148) (SEQ ID NO: 149) (SEQ ID NO: 150) H VH NYDMS
FINTVGYAYYASWA LDNYYTWGI (SEQ ID NO: 151) KG (SEQ ID NO: 153) (SEQ
ID NO: 152) I VL QAAEDIYSSLA AASILAS QTNYGISSYGAA (SEQ ID NO: 154)
(SEQ ID NO: 155) (SEQ ID NO: 156) I VH NKPIT WISTTGSAYYASWA
YSSDYGHHDL (SEQ ID NO: 157) KG (SEQ ID NO: 159) (SEQ ID NO: 158) J
VL QASQSVYRDYLS GASNLAS LGGFSGNINT (SEQ ID NO: 160) (SEQ ID NO:
161) (SEQ ID NO: 162) J VH NYHMC IISNTGYTYYSSWAK DRLANL (SEQ ID NO:
163) G (SEQ ID NO: 165) (SEQ ID NO: 164) K VL QASQSISTYLK SASTLTS
QSNAGSSSSSCGYA (SEQ ID NO: 166) (SEQ ID NO: 167) (SEQ ID NO: 168) K
VH YYGMG IISGIGNTYYPTWAK GDF (SEQ ID NO: 169) G (SEQ ID NO: 171)
(SEQ ID NO: 170) L VL QASQSISRYLK SASTLTS QSNAGSSSSSCGYA (SEQ ID
NO: 172) (SEQ ID NO: 173) (SEQ ID NO: 174) L VH YYGMG
IISGSGNTYYASWAK GDF (SEQ ID NO: 175) G (SEQ ID NO: 177) (SEQ ID NO:
176) M VL QSSQSVYSNYLS VASSLAS GGFQKYIDDGGA (SEQ ID NO: 178) (SEQ
ID NO: 179) (SEQ ID NO: 180) M VH TYAMS IMSSSGSAYYASWAK SSSFGL (SEQ
ID NO: 181) G (SEQ ID NO: 183) (SEQ ID NO: 182) N VK SVSSSISSTNLH
GTSNLAS QQWSTTYT (SEQ ID NO: 184) (SEQ ID NO: 185) (SEQ ID NO: 186)
N VH DYYIH RIDPDNGESTYVPKF EGLDYGDYYAVDY (SEQ ID NO: 187) QG (SEQ
ID NO: 189) (SEQ ID NO: 188) O VK RASESVDSYGNSFMH RASNLES QQNYEDPLT
(SEQ ID NO: 190) (SEQ ID NO: 191) (SEQ ID NO: 192) O VH NYLIE
VINPGSGIINYNEKFK DWDTFYSYEREVY (SEQ ID NO: 193) I AMDY (SEQ ID NO:
194) (SEQ ID NO: 195) P VK RASQSIGTNIH FASESIS QQSNSWPLT (SEQ ID
NO: 196) (SEQ ID NO: 197) (SEQ ID NO: 198) P VH DYYVH
WIDPENGDTEYAPKF GYGNFYFDY (SEQ ID NO: 199) QG (SEQ ID NO: 201) (SEQ
ID NO: 200) Q VK KASQDVGTAVA WASTRHT QQYSSYPYT (SEQ ID NO: 202)
(SEQ ID NO: 203) (SEQ ID NO: 204) Q VH DYFMH WIDPENGDTEYAPKFQG
GQATYYFDY (SEQ ID NO: 205) (SEQ ID NO: 206) (SEQ ID NO: 207) R VK
HATQNINVWLS KTSNLHT QQGQSFPYT (SEQ ID NO: 208) (SEQ ID NO: 209)
(SEQ ID NO: 210) R VH DYFMH WIDPENGDTEYAPKFQG GQATYYFDY (SEQ ID NO:
211) (SEQ ID NO: 212) (SEQ ID NO: 213) S VK KSSQSLLYSNNQKNYLA
WASTRES QQYYDYPWT (SEQ ID NO: 214) (SEQ ID NO: 215) (SEQ ID NO:
216) S VH DYFMH WIDPENGDTEYAPKF GQATYYFDY (SEQ ID NO: 217) (SEQ ID
NO: 218)QG SEQ ID NO: 219) T VK RTSQDINNFLN HTSRLKS QHYYNLPWT (SEQ
ID NO: 220) (SEQ ID NO: 221) (SEQ ID NO: 222) T VH DNIMV
NINPYYGSPSYNLKFKD EGGNYGSLDN (SEQ ID NO: 223) (SEQ ID NO: 224) (SEQ
ID NO: 225) U VK KSSQSLLYSNNQKNYLA WASTRES QQYYDYPWT (SEQ ID NO:
226) (SEQ ID NO: 227) (SEQ ID NO: 228) U VH DNIMV NINPYYGSPSYNLKFKD
EGGNYGSLDN (SEQ ID NO: 229) (SEQ ID NO: 230) (SEQ ID NO: 231) V VK
RTSQDINNFLN HTSRLKS QHYYNLPWT (SEQ ID NO: 232) (SEQ ID NO: 233)
(SEQ ID NO: 234) V VH YYYMS TISIDGSTYYASWAEG GHINTGMDP (SEQ ID NO:
235) (SEQ ID NO: 236) (SEQ ID NO: 237) W VK RSSQTIVHSNGNTYLE
EVSNRFS FQGSHFPHT (SEQ ID NO: 238) (SEQ ID NO: 239) (SEQ ID NO:
240) W VH DYYMY WIDPENGDTECAPKFQG DRYDEGAASDYGMDY (SEQ ID NO: 241)
(SEQ ID NO: 242) (SEQ ID NO: 243) X VK RSSQSIVHSNGNTHLE KVSNRFS
FQGSHAPHT (SEQ ID NO: 244) (SEQ ID NO: 245) (SEQ ID NO: 246) X VH
DYYMY WIDPGNGDTECAPKFQG DRYDEGAASDYAVDY (SEQ ID NO: 247) (SEQ ID
NO: 248) (SEQ ID NO: 249) Y VK RSSQTIVHSNGNTYLE EVSNRFS FQGSLFPHT
(SEQ ID NO: 250) (SEQ ID NO: 251) (SEQ ID NO: 252) Y VH DYYMY
WIDPENGDTECAPKFQG DRYDEGAASDYAVDY (SEQ ID NO: 253) (SEQ ID NO: 254)
(SEQ ID NO: 255)
The following table, Table 2, provides the SEQ ID NOs for the
antibody polynucleotides and polypeptides disclosed above, without
leader sequences.
TABLE-US-00006 TABLE 2 POLYPEPTIDES POLYNUCLEOTIDES SEQ ID NO SEQ
ID NO SEQ ID NO SEQ ID NO Antibody with leader without leader with
leader without leader A VK 2 256 3 257 A VH 4 258 5 259 B VK 6 260
7 261 B VH 8 262 9 263 C VK 10 264 11 265 C VH 12 266 13 267 D VK
14 268 15 269 D VH 16 270 17 271 E VK 18 272 19 273 E VH 20 274 21
275 F VK 22 276 23 277 F VH 24 278 25 279 G VK 26 280 27 281 G VH
28 282 29 283 H VK 30 284 31 285 H VH 32 286 33 287 I VK 34 288 35
289 I VH 36 290 37 291 J VK 38 292 39 293 J VH 40 294 41 295 K VK
42 296 43 297 K VH 44 298 45 299 L VK 46 300 47 301 L VH 48 302 49
303 M VK 50 304 51 305 M VH 52 306 53 307 N VK 56 308 57 309 N VH
58 310 59 311 O VK 60 312 61 313 O VH 62 314 63 315 P VK 64 316 65
317 P VH 66 318 67 319 Q VK 68 320 69 321 Q VH 70 322 71 323 R VK
72 324 73 325 R VH 74 326 75 327 S VK 76 328 77 329 S VH 78 330 79
331 T VK 80 332 81 333 T VH 82 334 83 335 U VK 84 336 85 337 U VH
86 338 87 339 V VK 88 340 89 341 V VH 90 342 91 343 W VK 92 344 93
345 W VH 94 346 95 347 X VK 96 348 97 349 X VH 98 350 99 351 Y VK
100 352 101 353 Y VH 102 354 103 355
[0066] An oligopeptide or polypeptide is within the scope of the
invention if it has an amino acid sequence that is at least 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to least one of the CDR's of Table 2 above; and/or to a CDR of a
sclerostin binding agent that cross-blocks the binding of at least
one of antibodies A-Y to sclerostin; and/or is cross-blocked from
binding to sclerostin by at least one of antibodies A-Y; and/or to
a CDR of a sclerostin binding agent wherein the binding agent can
block the inhibitory effect of sclerostin in a cell based
mineralization assay (i.e., a sclerostin neutralizing binding
agent); and/or to a CDR of a sclerostin binding agent that binds to
a Loop 2 epitope.
[0067] Sclerostin binding agent polypeptides and antibodies are
within the scope of the invention if they have amino acid sequences
that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to a variable region of at
least one of antibodies A-Y to sclerostin; and/or are cross-blocked
from binding to sclerostin by at least one of antibodies A-Y;
and/or can block the inhibitory effect of sclerostin in a cell
based mineralization assay (i.e., a sclerostin neutralizing binding
agent).
[0068] Polynucleotides encoding sclerostin binding agents are
within the scope of the invention if they have polynucleotide
sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide
encoding a variable region of at least one of antibodies A-Y, and
wherein the encoded sclerostin binding agents cross-block the
binding of at least one of antibodies A-Y to sclerostin; and/or are
cross-blocked from binding to sclerostin by at least one of
antibodies A-Y; and/or can block the inhibitory effect of
sclerostin in a cell based mineralization assay (i.e. a sclerostin
neutralizing binding agent); and/or bind to a Loop 2 epitope.
[0069] Antibodies according to the invention may have a binding
affinity for human sclerostin of less than or equal to
1.times.10.sup.-7M, less than or equal to 1.times.10.sup.-8M, less
than or equal to 1.times.10.sup.-9M, less than or equal to
1.times.10.sup.10 M, less than or equal to 1.times.10.sup.-11M, or
less than or equal to 1.times.10.sup.-12M.
[0070] The affinity of a binding agent such as an antibody or
binding partner, as well as the extent to which a binding agent
(such as an antibody) inhibits binding, can be determined by one of
ordinary skill in the art using conventional techniques, for
example, those described by Scatchard et al., Ann. N.Y. Acad. Sci.,
51:660-672 (1949) or by surface plasmon resonance (SPR; BIAcore,
Biosensor, Piscataway, N.J.). For surface plasmon resonance, target
molecules are immobilized on a solid phase and exposed to ligands
in a mobile phase running along a flow cell. If ligand binding to
the immobilized target occurs, the local refractive index changes,
leading to a change in SPR angle, which can be monitored in real
time by detecting changes in the intensity of the reflected light.
The rates of change of the SPR signal can be analyzed to yield
apparent rate constants for the association and dissociation phases
of the binding reaction. The ratio of these values gives the
apparent equilibrium constant (affinity) (see, e.g., Wolff et al.,
Cancer Res., 53:2560-65 (1993)).
[0071] An antibody according to the present invention may belong to
any immunoglobin class, for example IgG, IgE, IgM, IgD, or IgA. It
may be obtained from or derived from an animal, for example, fowl
(e.g., chicken) and mammals, which includes, but is not limited, to
a mouse, rat, hamster, rabbit, or other rodent, cow, horse, sheep,
goat, camel, human, or other primate. The antibody may be an
internalizing antibody. Production of antibodies is disclosed
generally in U.S. Patent Publication No. 2004/0146888 A1.
Characterization Assays
[0072] In the methods described above to generate antibodies
according to the invention, including the manipulation of the
specific antibody A-Y CDRs into new frameworks and/or constant
regions, appropriate assays are available to select the desired
antibodies or binding agents (i.e., assays for determining binding
affinity to sclerostin; cross-blocking assays; Biacore-based "human
sclerostin peptide epitope competition binding assay;" MC3T3-E1
cell based assay; and/or in vivo assays).
[0073] Cross-Blocking Assays
[0074] The terms "cross-block," "cross-blocked" and
"cross-blocking" are used interchangeably herein to mean the
ability of an antibody or other binding agent to interfere with the
binding of other antibodies or binding agents to sclerostin.
[0075] The extent to which an antibody or other binding agent is
able to interfere with the binding of another to sclerostin, and
therefore whether it can be said to cross-block according to the
invention, can be determined using competition binding assays. One
particularly suitable quantitative assay uses a Biacore machine
which can measure the extent of interactions using surface plasmon
resonance technology. Another suitable quantitative cross-blocking
assay uses an ELISA-based approach to measure competition between
antibodies or other binding agents in terms of their binding to
sclerostin.
[0076] Biacore Cross-Blocking Assay
[0077] The following generally describes a suitable Biacore assay
for determining whether an antibody or other binding agent
cross-blocks or is capable of cross-blocking according to the
invention. For convenience, reference is made to two antibodies,
but it will be appreciated that the assay can be used with any of
the sclerostin binding agents described herein. The Biacore machine
(for example the Biacore 3000) is operated in line with the
manufacturer's recommendations.
[0078] Thus, in one cross-blocking assay, sclerostin is coupled to
a CM5 Biacore chip using standard amine coupling chemistry to
generate a sclerostin-coated surface. Typically 200-800 resonance
units of sclerostin would be coupled to the chip (an amount that
gives easily measurable levels of binding but that is readily
saturable by the concentrations of test reagent being used).
[0079] The two antibodies (termed 1* and 2*) to be assessed for
their ability to cross-block each other are mixed at a one to one
molar ratio of binding sites in a suitable buffer to create the
test mixture. When calculating the concentrations on a binding site
basis the molecular weight of an antibody is assumed to be the
total molecular weight of the antibody divided by the number of
sclerostin binding sites on that antibody.
[0080] The concentration of each antibody in the test mix should be
high enough to readily saturate the binding sites for that antibody
on the sclerostin molecules captured on the Biacore chip. The
antibodies in the mixture are at the same molar concentration (on a
binding basis) and that concentration would typically be between
1.00 and 1.5 micromolar (on a binding site basis).
[0081] Separate solutions containing antibody 1* alone and antibody
2* alone are also prepared. Antibody 1* and antibody 2* in these
solutions should be in the same buffer and at the same
concentration as in the test mix.
[0082] The test mixture is passed over the sclerostin-coated
Biacore chip and the total amount of binding recorded. The chip is
then treated in such a way as to remove the bound antibodies
without damaging the chip-bound sclerostin. Typically this is done
by treating the chip with 30 mM HCl for 60 seconds.
[0083] The solution of antibody 1* alone is then passed over the
sclerostin-coated surface and the amount of binding recorded. The
chip is again treated to remove all of the bound antibody without
damaging the chip-bound sclerostin.
[0084] The solution of antibody 2* alone is then passed over the
sclerostin-coated surface and the amount of binding recorded.
[0085] The maximum theoretical binding of the mixture of antibody
1* and antibody 2* is next calculated, and is the sum of the
binding of each antibody when passed over the sclerostin surface
alone. If the actual recorded binding of the mixture is less than
this theoretical maximum then the two antibodies are cross-blocking
each other.
[0086] Thus, in general, a cross-blocking antibody or other binding
agent according to the invention is one which will bind to
sclerostin in the above Biacore cross-blocking assay such that
during the assay and in the presence of a second antibody or other
binding agent of the invention the recorded binding is between 80%
and 0.1% (e.g., between 80% and 4%) of the maximum theoretical
binding, specifically between 75% and 0.1% (e.g., between 75% and
4%) of the maximum theoretical binding, and more specifically
between 70% and 0.1% (e.g., between 70% and 4%) of maximum
theoretical binding (as just defined above) of the two antibodies
or binding agents in combination.
[0087] The Biacore assay described above is a primary assay used to
determine if antibodies or other binding agents cross-block each
other according to the invention. On rare occasions particular
antibodies or other binding agents may not bind to sclerostin
coupled via amine chemistry to a CM5 Biacore chip (this usually
occurs when the relevant binding site on sclerostin is masked or
destroyed by the coupling to the chip). In such cases
cross-blocking can be determined using a tagged version of
Sclerostin, for example N-terminal His-tagged Sclerostin (R & D
Systems, Minneapolis, Minn., USA; 2005 cat#1406-ST-025). In this
particular format, an anti-His antibody would be coupled to the
Biacore chip and then the His-tagged sclerostin would be passed
over the surface of the chip and captured by the anti-His antibody.
The cross blocking analysis would be carried out essentially as
described above, except that after each chip regeneration cycle,
new His-tagged sclerostin would be loaded back onto the anti-His
antibody coated surface. In addition to the example given using
N-terminal His-tagged sclerostin, C-terminal His-tagged sclerostin
could alternatively be used. Furthermore, various other tags and
tag binding protein combinations that are known in the art could be
used for such a cross-blocking analysis (e.g., HA tag with anti-HA
antibodies; FLAG tag with anti-FLAG antibodies; and/or biotin tag
with streptavidin).
ELISA-Based Cross-Blocking Assay
[0088] The following generally describes an ELISA assay for
determining whether an anti-sclerostin antibody or other sclerostin
binding agent cross-blocks or is capable of cross-blocking
according to the invention. For convenience, reference is made to
two antibodies (Ab-1 and Ab-2), but it will be appreciated that the
assay can be used with any of the sclerostin binding agents
described herein.
[0089] The general principle of the assay is to have an
anti-sclerostin antibody coated onto the wells of an ELISA plate.
An excess amount of a second, potentially cross-blocking,
anti-sclerostin antibody is added in solution (i.e., not bound to
the ELISA plate). A limited amount of sclerostin is then added to
the wells. The coated antibody and the antibody in solution compete
for binding of the limited number of sclerostin molecules. The
plate is washed to remove sclerostin that has not been bound by the
coated antibody and to also remove the second, solution phase
antibody as well as any complexes formed between the second,
solution phase antibody and sclerostin. The amount of bound
sclerostin is then measured using an appropriate sclerostin
detection reagent. An antibody in solution that is able to
cross-block the coated antibody will be able to cause a decrease in
the number of sclerostin molecules that the coated antibody can
bind relative to the number of sclerostin molecules that the coated
antibody can bind in the absence of the second, solution phase,
antibody.
[0090] This assay is described below in more detail for Ab-1 and
Ab-2. In the instance where Ab-1 is chosen to be the immobilized
antibody, it is coated onto the wells of the ELISA plate, after
which the plates are blocked with a suitable blocking solution to
minimize non-specific binding of reagents that are subsequently
added. An excess amount of Ab-2 is then added to the ELISA plate
such that the moles of Ab-2 sclerostin binding sites per well are
at least 10 fold higher than the moles of Ab-1 sclerostin binding
sites that were used, per well, during the coating of the ELISA
plate. Sclerostin is then added such that the moles of sclerostin
added per well are at least 25-fold lower than the moles of Ab-1
sclerostin binding sites that were used for coating each well.
Following a suitable incubation period, the ELISA plate is washed
and a sclerostin detection reagent is added to measure the amount
of sclerostin specifically bound by the coated anti-sclerostin
antibody (in this case Ab-1). The background signal for the assay
is defined as the signal obtained in wells with the coated antibody
(in this case Ab-1), second solution phase antibody (in this case
Ab-2), sclerostin buffer only (i.e., no sclerostin) and sclerostin
detection reagents. The positive control signal for the assay is
defined as the signal obtained in wells with the coated antibody
(in this case Ab-1), second solution phase antibody buffer only
(i.e., no second solution phase antibody), sclerostin and
sclerostin detection reagents. The ELISA assay needs to be run in
such a manner so as to have the positive control signal be at least
6 times the background signal.
[0091] To avoid any artifacts resulting from the choice of which
antibody to use as the coating antibody and which to use as the
second (competitor) antibody (e.g., significantly different
affinities between Ab-1 and Ab-2 for sclerostin), the
cross-blocking assay can be run in two formats: [0092] 1) format 1
is where Ab-1 is the antibody that is coated onto the ELISA plate
and Ab-2 is the competitor antibody that is in solution and [0093]
2) format 2 is where Ab-2 is the antibody that is coated onto the
ELISA plate and Ab-1 is the competitor antibody that is in
solution.
[0094] Ab-1 and Ab-2 are defined as cross-blocking if, either in
format 1 or in format 2, the solution phase anti-sclerostin
antibody is able to cause a reduction of between 60% and 100%,
specifically between 70% and 100%, and more specifically between
80% and 100%, of the sclerostin detection signal (i.e., the amount
of sclerostin bound by the coated antibody) as compared to the
sclerostin detection signal obtained in the absence of the solution
phase anti-sclerostin antibody (i.e., the positive control
wells).
[0095] An example of such an ELISA-based cross blocking assay can
be found in Example 3 ("ELISA-based cross-blocking assay").
Cell-Based Neutralization Assay
[0096] Mineralization by osteoblast-lineage cells in culture,
either primary cells or cell lines, is used as an in vitro model of
bone formation. Mineralization takes from about one to six weeks to
occur beginning with the induction of osteoblast-lineage cell
differentiation by one or more differentiation agents. The overall
sequence of events involves cell proliferation, differentiation,
extracellular matrix production, matrix maturation, and finally
deposition of mineral, which refers to crystallization and/or
deposition of calcium phosphate. This sequence of events starting
with cell proliferation and differentiation, and ending with
deposition of mineral, is referred to herein as mineralization.
Measurement of calcium (mineral) is the output of the assay.
[0097] MC3T3-E1 cells (Sudo et al. "In vitro differentiation and
calcification in a new clonal osteogenic cell line derived from
newborn mouse calvaria." J. Cell Biol., 96:191-198 (1983)) and
subclones of the original cell line can form mineral in culture
upon growth in the presence of differentiating agents. Such
subclones include MC3T3-E1-BF (Smith et al. "Glucocorticoids
inhibit developmental stage-specific osteoblast cell cycle." J.
Biol. Chem., 275:19992-20001 (2000)). For both the MC3T3-E1-BF
subclone as well as the original MC3T3-E1 cells, sclerostin can
inhibit one or more of the sequence of events leading up to and
including mineral deposition (i.e., sclerostin inhibits
mineralization). Anti-sclerostin antibodies that are able to
neutralize sclerostin's inhibitory activity allow for
mineralization of the culture in the presence of sclerostin such
that there is a statistically significant increase in deposition of
calcium phosphate (measured as calcium) as compared to the amount
of calcium measured in the sclerostin-only (i.e., no antibody)
treatment group.
[0098] When running the assay with the goal of determining whether
a particular anti-sclerostin antibody or anti-sclerostin binding
agent can neutralize sclerostin (i.e., is a sclerostin neutralizing
antibody or derivative thereof, or is a sclerostin neutralizing
binding agent), the amount of sclerostin used in the assay can be
the minimum amount of sclerostin that causes at least a 70%,
statistically significant, reduction in deposition of calcium
phosphate (measured as calcium) in the sclerostin-only group, as
compared to the amount of calcium measured in the no sclerostin
group. An anti-sclerostin neutralizing antibody or an
anti-sclerostin neutralizing binding agent is defined as one that
causes a statistically significant increase in deposition of
calcium phosphate (measured as calcium) as compared to the amount
of calcium measured in the sclerostin-only (i.e., no antibody, no
binding agent) treatment group. To determine whether an
anti-sclerostin antibody or an anti-sclerostin binding agent is
neutralizing or not, the amount of anti-sclerostin antibody or
anti-sclerostin binding agent used in the assay needs to be such
that there is an excess of moles of sclerostin binding sites per
well as compared to the number of moles of sclerostin per well.
Depending on the potency of the antibody, the fold excess that may
be required can be 24, 18, 12, 6, 3, or 1.5, and one of skill is
familiar with the routine practice of testing more than one
concentration of binding agent. For example, a very potent
anti-sclerostin neutralizing antibody or anti-sclerostin
neutralizing binding agent will be able to neutralize sclerostin
even when there is less than a 6-fold excess of moles of sclerostin
binding sites per well as compared to the number of moles of
sclerostin per well. A less potent anti-sclerostin neutralizing
antibody or anti-sclerostin neutralizing binding agent will be able
to neutralize sclerostin only at a 12, 18, or 24 fold excess.
Sclerostin binding agents within this full range of potencies are
suitable as neutralizing sclerostin binding agents.
[0099] Anti-sclerostin antibodies and derivatives thereof that can
neutralize human sclerostin, and sclerostin binding agents that can
neutralize human sclerostin, may be of use in the treatment of
human conditions/disorders that are caused by, associated with, or
result in at least one of low bone formation, low bone mineral
density, low bone mineral content, low bone mass, low bone quality
and low bone strength.
In Vivo Neutralization Assay
[0100] Increases in various parameters associated with, or that
result from, the stimulation of new bone formation can be measured
as an output from in vivo testing of sclerostin binding agents in
order to identify those binding agents that are able to neutralize
sclerostin and thus able to cause stimulation of new bone
formation. Such parameters include various serum anabolic markers
(e.g., osteocalcin and P1NP (n-terminal propeptide of type 1
procollagen)), histomorphometric markers of bone formation (e.g.
osteoblast surface/bone surface; bone formation rate/bone surface;
and trabecular thickness), bone mineral density, bone mineral
content, bone mass, bone quality, and bone strength. A sclerostin
neutralizing binding agent is defined as one capable of causing a
statistically significant increase, as compared to vehicle treated
animals, in any parameter associated with, or that results from,
the stimulation of new bone formation. Such in vivo testing can be
performed in any suitable mammal (e.g. mouse, rat, and/or
monkey).
[0101] Although the amino acid sequence of sclerostin is not 100%
identical across mammalian species (e.g., mouse sclerostin is not
100% identical to human sclerostin), it will be appreciated by one
skilled in the art that a sclerostin binding agent that can
neutralize, in vivo, the sclerostin of a certain species (e.g.,
mouse) and that also can bind human sclerostin in vitro is very
likely to be able to neutralize human sclerostin in vivo. Thus,
such a human sclerostin binding agent (e.g., anti-human sclerostin
antibody) may be of use in the treatment of human
conditions/disorders that are caused by, associated with, or result
in at least one of low bone formation, low bone mineral density,
low bone mineral content, low bone mass, low bone quality, and low
bone strength. Mice in which homologous recombination had been used
to delete the mouse sclerostin gene and insert the human sclerostin
gene in its place (i.e., human sclerostin gene knock-in mice or
human SOST knock-in mice) would be an example of an additional in
vivo system.
[0102] Pharmaceutical compositions are provided, comprising one of
the above-described binding agents such as at least one of antibody
A-Y to human sclerostin, along with a pharmaceutically or
physiologically acceptable carrier, excipient, or diluent.
Pharmaceutical compositions and methods of treatment are disclosed
in copending application Ser. No. 10/868,497, filed Jun. 16, 2004,
published as U.S. 2005/0106683, which claims priority to Ser. No.
60/478,977, both of which are incorporated by reference herein.
[0103] The development of suitable dosing and treatment regimens
for using the particular compositions described herein in a variety
of treatment regimens, including e.g., subcutaneous, oral,
parenteral, intravenous, intranasal, and intramuscular
administration and formulation, is well known in the art, some of
which are briefly discussed below for general purposes of
illustration.
[0104] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to an
animal. As such, these compositions may be formulated with an inert
diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0105] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein subcutaneously,
parenterally, intravenously, intramuscularly, or intraperitoneally.
Such approaches are well known to the skilled artisan, some of
which are further described, for example, in U.S. Pat. No.
5,543,158; U.S. Pat. No. 5,641,515; and U.S. Pat. No. 5,399,363. In
certain embodiments, solutions of the active compounds as free base
or pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will
contain a preservative to prevent the growth of microorganisms.
[0106] Illustrative pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (for example, see U.S. Pat. No.
5,466,468). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion, and/or by the use of surfactants. The
prevention of the action of microorganisms can be facilitated by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0107] In one embodiment, for parenteral administration in an
aqueous solution, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, a sterile aqueous medium that can be employed will be
known to those of skill in the art. For example, one dosage may be
dissolved in 1 ml of isotonic NaCl solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site
of infusion (see, for example, Remington's Pharmaceutical Sciences,
15th ed., pp. 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. Moreover, for human administration, preparations
will preferably meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologics
standards.
[0108] In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form.
Illustrative pharmaceutically-acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine, and the like. Upon
formulation, solutions will be administered in a manner compatible
with the dosage formulation and in such amount as is
therapeutically effective.
[0109] The carriers can further comprise any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human.
[0110] In certain embodiments, liposomes, nanocapsules,
microparticles, lipid particles, vesicles, and the like, are used
for the introduction of the compositions of the present invention
into suitable host cells/organisms. In particular, the compositions
of the present invention may be formulated for delivery either
encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like. Alternatively,
compositions of the present invention can be bound, either
covalently or non-covalently, to the surface of such carrier
vehicles.
[0111] The formation and use of liposome and liposome-like
preparations as potential drug carriers is generally known to those
of skill in the art (see for example, Lasic, Trends Biotechnol.,
16(7):307-21 (1998); Takakura, Nippon Rinsho, 56(3):691-95 (1998);
Chandran et al., Indian J. Exp. Biol., 35(8):801-09 (1997);
Margalit, Crit. Rev. Ther. Drug Carrier Syst., 12(2-3):233-61
(1995); U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat.
No. 5,565,213; U.S. Pat. No. 5,738,868, and U.S. Pat. No.
5,795,587, each specifically incorporated herein by reference in
its entirety). The use of liposomes does not appear to be
associated with autoimmune responses or unacceptable toxicity after
systemic delivery. In certain embodiments, liposomes are formed
from phospholipids that are dispersed in an aqueous medium and
spontaneously form multilamellar concentric bilayer vesicles (also
termed multilamellar vesicles (MLVs)).
[0112] Alternatively, in other embodiments, the invention provides
for pharmaceutically-acceptable nanocapsule formulations of the
compositions of the present invention. Nanocapsules can generally
entrap compounds in a stable and reproducible way (see, for
example, Quintanar-Guerrero et al., Drug Dev. Ind. Pharm.,
24(12):1113-28 (1998)). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) may be designed using polymers able to be degraded in vivo.
Such particles can be made as described, for example, by Couvreur
et al., Crit. Rev. Ther. Drug Carrier Syst., 5(1):1-20 (1988); zur
Muhlen et al., Eur. J. Pharm. Biopharm., 45(2):149-55 (1998);
Zambaux et al., J. Controlled Release, 50(1-3):31-40 (1998); and
U.S. Pat. No. 5,145,684.
[0113] In addition, pharmaceutical compositions of the present
invention may be placed within containers, along with packaging
material that provides instructions regarding the use of such
pharmaceutical compositions. Generally, such instructions will
include a tangible expression describing the reagent concentration,
as well as within certain embodiments, relative amounts of
excipient ingredients or diluents (e.g., water, saline or PBS) that
may be necessary to reconstitute the pharmaceutical
composition.
[0114] The dose administered may range from 0.01 mg/kg to 100 mg/kg
of body weight. As will be evident to one of skill in the art, the
amount and frequency of administration will depend, of course, on
such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient, and so
forth. Typically, the compositions may be administered by a variety
of techniques, as noted above.
[0115] Increases in bone mineral content and/or bone mineral
density may be determined directly through the use of X-rays (e.g.,
Dual Energy X-ray Absorptometry or "DEXA"), or by inference through
the measurement of (1) markers of bone formation and/or osteoblast
activity, such as, but not limited to, osteoblast specific alkaline
phosphatase, osteocalcin, type 1 procollagen C' propeptide (PICP),
total alkaline phosphatase (see Comier, Curr. Opin. in Rheu., 7:243
(1995)) and serum procollagen 1 N-terminal propeptide (P1NP) and/or
(2) markers of bone resorption and/or osteoclast activity
including, but not limited to, pyridinoline, deoxypryridinoline,
N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant
acid phosphatases, and galactosyl hydroxylysine (see Cornier, id),
serum TRAP 5b (tartrate-resistant acid phosphatase isoform 5b) and
serum cross-linked C-telopeptide (sCTXI). The amount of bone mass
may also be calculated from body weights or by using other methods
(see Guinness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)).
Animals and particular animal models are used in the art for
testing the effect of the compositions and methods of the invention
on, for example, parameters of bone loss, bone resorption, bone
formation, bone strength or bone mineralization that mimic
conditions of human disease such as osteoporosis and osteopenias.
Examples of such models include the ovariectomized rat model (Kalu,
D. N., "The ovariectomized rat model of postmenopausal bone loss."
Bone and Mineral, 15:175-192 (1991); Frost, H. M. and Jee, W. S.
S., "On the rat model of human osteopenias and osteoporosis." Bone
and Mineral, 18:227-236 (1992); and Jee, W. S. S, and Yao, W.,
"Overview: animal models of osteopenia and osteoporosis." J.
Musculoskel. Neuron. Interact., 1:193-207 (2001)).
[0116] Particular conditions which may be treated by the
compositions of the present invention include dysplasias, wherein
growth or development of bone is abnormal and a wide variety of
causes of osteopenia, osteoporosis, and bone loss. Representative
examples of such conditions include achondroplasia, cleidocranial
dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's Disease,
hypophosphatemic rickets, Marfan's syndrome, multiple hereditary
exotoses, neurofibromatosis, osteogenesis imperfecta,
osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis,
and pyogenic osteomyelitis, periodontal disease, anti-epileptic
drug induced bone loss, primary and secondary hyperparathyroidism,
familial hyperparathyroidism syndromes, weightlessness induced bone
loss, osteoporosis (e.g., osteoporosis in men), postmenopausal bone
loss, osteoarthritis, renal osteodystrophy, infiltrative disorders
of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's
disease, melorheostosis, metabolic bone diseases, mastocytosis,
sickle cell anemia/disease, organ transplant related bone loss,
kidney transplant related bone loss, systemic lupus erythematosus,
ankylosing spondylitis, epilepsy, juvenile arthritides,
thalassemia, mucopolysaccharidoses, fabry disease, Turner syndrome,
Down Syndrome, Klinefelter Syndrome, leprosy, Perthes' Disease,
adolescent idiopathic scoliosis, infantile onset multi-system
inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's
Disease, ischemic bone disease (such as Legg-Calve-Perthes disease,
regional migratory osteoporosis), anemic states, conditions caused
by steroids, glucocorticoid-induced bone loss, heparin-induced bone
loss, bone marrow disorders, scurvy, malnutrition, calcium
deficiency, idiopathic osteopenia or osteoporosis, congenital
osteopenia or osteoporosis, alcoholism, chronic liver disease,
postmenopausal state, chronic inflammatory conditions, rheumatoid
arthritis, inflammatory bowel disease, ulcerative colitis,
inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea,
pregnancy, diabetes mellitus, hyperthyroidism, thyroid disorders,
parathyroid disorders, Cushing's disease, acromegaly, hypogonadism,
immobilization or disuse, reflex sympathetic dystrophy syndrome,
regional osteoporosis, osteomalacia, bone loss associated with
joint replacement, HIV associated bone loss, bone loss associated
with loss of growth hormone, bone loss associated with cystic
fibrosis, fibrous dysplasia, chemotherapy associated bone loss,
tumor induced bone loss, cancer-related bone loss, hormone ablative
bone loss, multiple myeloma, drug-induced bone loss, anorexia
nervosa, disease associated facial bone loss, disease associated
cranial bone loss, disease associated bone loss of the jaw, disease
associated bone loss of the skull, and bone loss associated with
space travel. Further conditions relate to bone loss associated
with aging, including facial bone loss associated with aging,
cranial bone loss associated with aging, jaw bone loss associated
with aging, and skull bone loss associated with aging.
[0117] Compositions of the present invention may also be useful for
improving outcomes in orthopedic procedures, dental procedures,
implant surgery, joint replacement, bone grafting, bone cosmetic
surgery and bone repair such as fracture healing, nonunion healing,
delayed union healing and facial reconstruction. One or more
compositions may be administered before, during and/or after the
procedure, replacement, graft, surgery or repair.
[0118] The invention also provides a diagnostic kit comprising at
least one anti-sclerostin binding agent according to the present
invention. The binding agent may be an antibody. In addition, such
a kit may optionally comprise one or more of the following: [0119]
(1) instructions for using the one or more binding agent(s) for
screening, diagnosis, prognosis, therapeutic monitoring or any
combination of these applications; [0120] (2) a labeled binding
partner to the anti-sclerostin binding agent(s); [0121] (3) a solid
phase (such as a reagent strip) upon which the anti-sclerostin
binding agent(s) is immobilized; and [0122] (4) a label or insert
indicating regulatory approval for screening, diagnostic,
prognostic or therapeutic use or any combination thereof. If no
labeled binding partner to the binding agent(s) is provided, the
binding agent(s) itself can be labeled with one or more of a
detectable marker(s), e.g., a chemiluminescent, enzymatic,
fluorescent, or radioactive moiety.
[0123] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Recombinant Expression of Sclerostin
[0124] Recombinant human sclerostin/SOST is commercially available
from R&D Systems (Minneapolis, Minn., USA; 2006
cat#1406-ST-025). Additionally, recombinant mouse sclerostin/SOST
is commercially available from R&D Systems (Minneapolis, Minn.,
USA; 2006 cat#1589-ST-025).
[0125] Alternatively, the different species of sclerostin can be
expressed transiently in serum-free suspension adapted 293T or
293EBNA cells. Transfections can be performed as 500 mL or 1 L
cultures. The following reagents and materials are available from
Gibco BRL (now Invitrogen, Carlsbad, Calif.). Catalog numbers are
listed in parentheses: serum-free DMEM (21068-028); DMEM/F12 (3:1)
(21068/11765); 1.times. Insulin-Transferrin-Selenium Supplement
(51500-056); 1.times. Pen Strep Glut (10378-016); 2 mM 1-Glutamine
(25030-081); 20 mM HEPES (15630-080); 0.01% Pluronic F68
(24040-032). Briefly, the cell inoculum (5.0-10.0.times.10.sup.5
cells/mL.times.culture volume) is centrifuged at 2,500 RPM for 10
minutes at 4.degree. C. to remove the conditioned medium.
[0126] The cells are resuspended in serum-free DMEM and centrifuged
again at 2,500 RPM for 10 minutes at 4.degree. C. After aspirating
the wash solution, the cells are resuspended in growth medium
[DMEM/F12 (3:1)+1.times. Insulin-Transferrin-Selenium
Supplement+1.times. Pen Strep Glut+2 mM L-Glutamine+20 mM
HEPES+0.01% Pluronic F68] in a 1 L or 3 L spinner flask culture.
The spinner flask culture is maintained on magnetic stir plate at
125 RPM which is placed in a humidified incubator maintained at
37.degree. C. and 5% CO.sub.2. The mammalian expression plasmid DNA
(e.g., pcDNA3.1, pCEP4, Invitrogen Life Technologies, Carlsbad,
Calif.), containing the complete coding region (and stop codon) of
sclerostin with a Kozak consensus sequence (e.g., CCACC) directly
5' of the start site ATG, is complexed to the transfection reagent
in a 50 mL conical tube.
[0127] The DNA-transfection reagent complex can be prepared in
5-10% of the final culture volume in serum-free DMEM or OPTI-MEM.
The transfection reagents that can be used for this purpose include
X-tremeGene RO-1539 (Roche Applied Science, Indianapolis, Ind.),
FuGene6 (Roche Applied Science, Indianapolis, Ind.), Lipofectamine
2000 (Invitrogen, Carlsbad, Calif.), and 293fectin.TM. (Invitrogen,
Carlsbad, Calif.). 1-5 .mu.g plasmid DNA/mL culture is first added
to serum-free DMEM, followed by 1-5 .mu.l transfection reagent/mL
culture. The complexes can be incubated at room temperature for
approximately 10-30 minutes and then added to the cells in the
spinner flask. The transfection/expression can be performed for 4-7
days, after which the conditioned medium (CM) is harvested by
centrifugation at 4,000 RPM for 60 minutes at 4.degree. C.
Example 2
Purification of Recombinant Sclerostin
[0128] Recombinant sclerostin was purified from mammalian host
cells as follows. All purification processes were carried out at
room temperature. One purification scheme was used to purify
various species of sclerostin, including murine and human
sclerostin. The purification scheme used affinity chromatography
followed by cation exchange chromatography.
Heparin Chromatography
[0129] The mammalian host cell conditioned medium (CM) was
centrifuged in a Beckman J6-M1 centrifuge at 4000 rpm for 1 hour at
4.degree. C. to remove cell debris. The CM supernatant was then
filtered through a sterile 0.2 .mu.M filter. (At this point the
sterile filtered CM may be optionally stored frozen until
purification.) If the CM was frozen, it was thawed at the following
temperatures, or combination thereof: 4.degree. C., room
temperature or warm water. Following thawing, the CM was filtered
through a sterile 0.2 .mu.m filter and optionally concentrated by
tangential flow ultrafiltration (TFF) using a 10 kD molecular
weight cut-off membrane. The CM concentrate was filtered through a
sterile 0.2 .mu.m filter and then loaded onto a Heparin High
Performance (Heparin HP) column (GE Healthcare, formerly Amersham
Biosciences) equilibrated in PBS. Alternatively, the filtered CM
supernatant may be loaded directly onto the Heparin HP column
equilibrated in PBS.
[0130] After loading, the Heparin HP column was washed with PBS
until the absorbance at 280 nm of the flow-through returned to
baseline (i.e., absorbance measured before loading CM supernatant).
The sclerostin was then eluted from the column using a linear
gradient from 150 mM to 2M sodium chloride in PBS. The absorbance
at 280 nm of the eluate was monitored and fractions containing
protein were collected. The fractions were then assayed by
Coomassie-stained SDS-PAGE to identify fractions containing a
polypeptide that migrates at the size of glycosylated sclerostin.
The appropriate fractions from the column were combined to make the
Heparin HP pool.
Cation Exchange Chromatography
[0131] The sclerostin eluted from the Heparin HP column was further
purified by cation exchange chromatography using SP High
Performance (SPHP) chromatography media (GE Healthcare, formerly
Amersham Biosciences). The Heparin HP pool was buffer exchanged
into PBS by dialysis using 10,000 MWCO membranes (Pierce
Slide-A-Lyzer). The dialyzed Heparin HP pool was then loaded onto
an SPHP column equilibrated in PBS. After loading, the column was
washed with PBS until the absorbance at 280 nm of the flow-through
returned to baseline. The sclerostin was then eluted from the SPHP
column using a linear gradient from 150 mM to 1 M sodium chloride
in PBS. The absorbance at 280 nm of the eluate was monitored and
the eluted sclerostin was collected in fractions. The fractions
were then assayed by Coomassie-stained SDS-PAGE to identify
fractions containing a polypeptide that migrates at the size of
glycosylated sclerostin. The appropriate fractions from the column
were combined to make the SPHP pool.
Formulation
[0132] Following purification, the SPHP pool was formulated in PBS
by dialysis using 10,000 MWCO membranes (Pierce Slide-A-Lyzer). If
concentration of sclerostin was necessary, a centrifugal device
(Amicon Centricon or Centriprep) with a 10,000 MWCO membrane was
used. Following formulation the sclerostin was filtered through a
sterile 0.2 .mu.m filter and stored at 4.degree. C. or frozen.
Example 3
ELISA-Based Cross-Blocking Assay
[0133] An antibody is coated on an ELISA plate at 2 .mu.g/ml. While
plates are blocking, the test antibody is incubated with human
sclerostin at a final concentration of 25 ng/ml for one hour at
room temperature in a separate plate. This complex is then
transferred to the blocked ELISA plate and incubated for a further
one hour at room temperature. Plates are washed and a pool of
biotinylated anti-sclerostin antibodies at 1 ug/ml is then added
and incubated for one hour at room temperature. Plates are then
washed and streptavidin-horseradish peroxidase conjugate added at a
1:5000 dilution. Plates are developed with TMB. Blocking antibodies
are able to reduce the ELISA signal due to inhibition of sclerostin
binding to the coated antibodies. Positive crossblocking wells are
considered to be those wells which decrease the signal by at least
40%.
Example 4
ELISA-Based Cross-Blocking Assay
[0134] Liquid volumes used in this example would be those typically
used in 96-well plate ELISAs (e.g. 50-200 Ab-X and Ab-Y, in this
example are assumed to have molecular weights of about 145 Kd and
to have 2 sclerostin binding sites per antibody molecule. An
anti-sclerostin antibody (Ab-X) is coated (e.g. 50.mu. of 1
.mu.g/ml) onto a 96-well ELISA plate [e.g. Corning 96 Well EIA/RIA
Flat Bottom Microplate (Product #3590), Corning Inc., Acton, Mass.]
for at least one hour. After this coating step the antibody
solution is removed, the plate is washed once or twice with wash
solution (e.g., PBS and 0.05% Tween 20) and is then blocked using
an appropriate blocking solution (e.g., PBS, 1% BSA, 1% goat serum
and 0.5% Tween 20) and procedures known in the art. Blocking
solution is then removed from the ELISA plate and a second
anti-sclerostin antibody (Ab-Y), which is being tested for it's
ability to cross-block the coated antibody, is added in excess
(e.g. 50 .mu.l of 10 .mu.g/ml) in blocking solution to the
appropriate wells of the ELISA plate. Following this, a limited
amount (e.g. 50 .mu.l of 10 ng/ml) of sclerostin in blocking
solution is then added to the appropriate wells and the plate is
incubated for at least one hour at room temperature while shaking.
The plate is then washed 2-4 times with wash solution. An
appropriate amount of a sclerostin detection reagent [e.g.,
biotinylated anti-sclerostin polyclonal antibody that has been
pre-complexed with an appropriate amount of a
streptavidin-horseradish peroxidase (HRP) conjugate] in blocking
solution is added to the ELISA plate and incubated for at least one
hour at room temperature. The plate is then washed at least 4 times
with wash solution and is developed with an appropriate reagent
[e.g. HRP substrates such as TMB (colorimetric) or various HRP
luminescent substrates]. The background signal for the assay is
defined as the signal obtained in wells with the coated antibody
(in this case Ab-X), second solution phase antibody (in this case
Ab-Y), sclerostin buffer only (i.e. no sclerostin) and sclerostin
detection reagents. The positive control signal for the assay is
defined as the signal obtained in wells with the coated antibody
(in this case Ab-X), second solution phase antibody buffer only
(i.e. no second solution phase antibody), sclerostin and sclerostin
detection reagents. The ELISA assay needs to be run in such a
manner so as to have the positive control signal be at least 6
times the background signal.
[0135] To avoid any artifacts (e.g. significantly different
affinities between Ab-X and Ab-Y for sclerostin) resulting from the
choice of which antibody to use as the coating antibody and which
to use as the second (competitor) antibody, the cross-blocking
assay needs to be run in two formats:
[0136] 1) format 1 is where Ab-X is the antibody that is coated
onto the ELISA plate and Ab-Y is the competitor antibody that is in
solution and
[0137] 2) format 2 is where Ab-Y is the antibody that is coated
onto the ELISA plate and Ab-X is the competitor antibody that is in
solution.
[0138] Ab-X and Ab-Y are defined as cross-blocking if, either in
format 1 or in format 2, the solution phase anti-sclerostin
antibody is able to cause a reduction of between 60% and 100%,
specifically between 70% and 100%, and more specifically between
80% and 100%, of the sclerostin detection signal (i.e. the amount
of sclerostin bound by the coated antibody) as compared to the
sclerostin detection signal obtained in the absence of the solution
phase anti-sclerostin antibody (i.e. the positive control
wells).
[0139] In the event that a tagged version of sclerostin is used in
the ELISA, such as a N-terminal His-tagged Sclerostin (R&D
Systems, Minneapolis, Minn., USA; 2005 cat#1406-ST-025) then an
appropriate type of sclerostin detection reagent would include an
HRP labeled anti-His antibody. In addition to using N-terminal
His-tagged Sclerostin, one could also use C-terminal His-tagged
Sclerostin. Furthermore, various other tags and tag binding protein
combinations that are known in the art could be used in this
ELISA-based cross-blocking assay (e.g., HA tag with anti-HA
antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with
streptavidin).
[0140] From the foregoing, although specific embodiments of the
invention have been described herein for purposes of illustration,
various modifications may be made without deviating from the spirit
and scope of the invention. Accordingly, the invention is not
limited except as by the appended claims. All publications,
published patent applications, and patent documents disclosed
herein are hereby incorporated by reference.
Sequence CWU 1
1
2561190PRTHomo sapiens 1Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr
Glu Ile Ile Pro Glu 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro
Glu Leu Glu Asn Asn Lys Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly
Gly Arg Pro Pro His His Pro Phe Glu 35 40 45 Thr Lys Asp Val Ser
Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg 50 55 60 Tyr Val Thr
Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu 65 70 75 80 Val
Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile 85 90
95 Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile
100 105 110 Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro
Gly Gly 115 120 125 Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala
Ser Cys Lys Cys 130 135 140 Lys Arg Leu Thr Arg Phe His Asn Gln Ser
Glu Leu Lys Asp Phe Gly 145 150 155 160 Thr Glu Ala Ala Arg Pro Gln
Lys Gly Arg Lys Pro Arg Pro Arg Ala 165 170 175 Arg Ser Ala Lys Ala
Asn Gln Ala Glu Leu Glu Asn Ala Tyr 180 185 190 2134PRTOryctolagus
cuniculus 2Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu
Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln
Thr Pro Ser Pro 20 25 30 Val Ser Ala Gly Val Gly Gly Thr Val Thr
Ile Lys Cys Gln Ser Ser 35 40 45 Gln Arg Val Phe Asn Asn Asn Glu
Leu Ser Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Arg
Leu Ile Tyr Glu Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr
Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110
Leu Gly Gly Tyr Asp Asp Asp Gly Asp Asn Ala Phe Gly Gly Gly Thr 115
120 125 Glu Val Val Val Lys Arg 130 3402DNAOryctolagus cuniculus
3atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgcag ccgtgctgac ccagacacca tcgcccgtgt ctgcaggtgt gggaggcaca
120gtcaccatca agtgccagtc cagtcagagg gtttttaata acaacgaatt
atcctggtat 180cagcagaaac cagggcagcc gcccaaacgc ctgatctatg
aagcatccac actggcatct 240ggggtcccag ataggttcag cggcagtgga
tctgggacac agttcactct caccatcagc 300ggcgtgcagt gtgacgatgc
tgccacttac tactgtctag gcggttatga tgatgatggt 360gataatgctt
tcggcggagg gaccgaggtg gtggtcaaac gt 4024143PRTOryctolagus cuniculus
4Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val
Thr 20 25 30 Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu 35 40 45 Asn Asn Tyr Tyr Met Thr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Leu Ile Tyr Pro Asp
Gly Ser Thr Phe Tyr Ala Asn 65 70 75 80 Trp Ala Glu Gly Arg Phe Thr
Ser Ser Lys Thr Ser Thr Thr Val Thr 85 90 95 Leu Lys Met Thr Ser
Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Glu
Gly Gly Ala Gly Asp Asn Thr Gly Thr Glu Tyr Tyr Tyr 115 120 125 Gly
Val Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140
5429DNAOryctolagus cuniculus 5atggagactg ggctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg gggtcgcctg
gtcacgcctg ggacacccct gacactcacc 120tgcacagcct ctggattctc
cctcaataat tactatatga cctgggtccg ccaggctcca 180gggaagggac
tggaatggat cggactcatt tatcctgatg gtagcacatt ctacgcgaac
240tgggcggaag gccgattcac cagctccaag acctcgacca cggtgactct
gaagatgacc 300agtccgacaa ccgaggacac ggccacctat ttctgtgcca
gagaaggtgg tgctggtgat 360aatactggta ccgaatatta ctacggcgtg
gacctctggg gccagggcac cctcgtcacc 420gtctcgagc 4296135PRTOryctolagus
cuniculus 6Met Asp Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu
Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ile Val Met Thr Gln
Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala Val Gly Asp Thr Val Thr
Ile Asn Cys Gln Ala Ser 35 40 45 Glu Asn Val Tyr Asp Lys Ser Ala
Leu Ser Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu
Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser
Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr
Ile Ser Asp Val Val Cys Ala Asp Ala Ala Thr Tyr Tyr Cys 100 105 110
Ala Gly Tyr Lys Ser Ser Ile Ile Asp Gly Thr Ala Phe Gly Gly Gly 115
120 125 Thr Asp Val Val Val Lys Gly 130 135 7405DNAOryctolagus
cuniculus 7atggacatga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcca tcgtgatgac ccagactcca tcttccgtgt ctgcagctgt
gggagataca 120gtcaccatca attgccaggc cagtgagaac gtttatgata
aaagcgcctt atcctggtat 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatc tggcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gatgtggtgt
gtgcggatgc tgccacttac tactgtgcag gatataaaag tagtattatt
360gatggtactg ctttcggcgg agggaccgac gtggtggtca aagga
4058132PRTOryctolagus cuniculus 8Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45 Asn Asn
Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Trp Ile Gly Ala Ile Gly Ala Gly Gly Asn Thr Tyr Tyr Ala Ser Trp 65
70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr
Val Asp 85 90 95 Leu Arg Met Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 100 105 110 Ala Asn Gly Asp Leu Pro Gly Gly Ile Trp
Gly Pro Gly Thr Leu Val 115 120 125 Thr Val Ser Leu 130
9396DNAOryctolagus cuniculus 9atggagacag gcctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggga cacccctgac actcacctgc 120acagtctctg gattctccct
cagtaacaat gcactgagct gggtccgcca ggctccaggg 180aaggggctgg
aatggatcgg ggccattggt gctggtggta acacatacta tgcgagctgg
240gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggtggatct
gagaatgacc 300agtccgacaa ccgaggacac ggccacctat ttctgtgcca
acggggacct gccaggtggc 360atctggggcc caggcaccct ggtcaccgtc tccata
39610136PRTOryctolagus cuniculus 10Met Asp Ile Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Asp Ile Val Met Thr Gln Thr Ala Ser 20 25 30 Pro Val Ser Ala
Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala 35 40 45 Ser Gln
Ser Ile Ser Pro Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60
Gln Arg Pro Lys Leu Leu Ile Tyr Asp Val Ser Lys Leu Ala Ser Gly 65
70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe
Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Ala Asp Gly Ala Thr
Tyr Tyr Cys Gln 100 105 110 Ser Tyr Tyr Gly Ile Asn Ser Asn Ser Tyr
Gly Asn Ile Phe Gly Gly 115 120 125 Gly Thr Glu Val Val Val Lys Gly
130 135 11408DNAOryctolagus cuniculus 11atggacataa gggcccccac
tcagctgctg gggctcctac tgctctggct cccaggtgcc 60agatgtgctg acattgtgat
gacccagact gcatcccccg tgtctgcagc tgtgggaggc 120acagtcacca
tcaattgcca ggccagtcag agcattagcc ctgcattagc ctggtatcag
180cagaaaccag ggcagcgtcc caagctcctg atctacgatg tatcgaaact
ggcatctggg 240gtcccatcgc ggttcagtgg cagtggatct gggacacagt
tcactctcac catcagcgac 300ctggagtgtg ccgatggtgc cacttactac
tgtcaaagct attatggtat taatagtaat 360agttatggta atattttcgg
cggagggacc gaggtggtgg tcaaagga 40812130PRTOryctolagus cuniculus
12Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu Ser 35 40 45 Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Tyr Ile Tyr Gly Asn Tyr
Asn Lys Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Thr Met Val Asp Leu 85 90 95 Lys Met Thr Ser Leu
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Gly
Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125 Ser
Ser 130 13390DNAOryctolagus cuniculus 13atggagacag gcctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtagctat gcaatgggct ggttccgcca ggctccaggg
180aaggggctgg agtggatcgg ctacatttat ggtaattata ataaatacta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccatgg
tggatctgaa gatgactagt 300ctgacaaccg aggacacggc cacctatttc
tgtgccagag ggggtgctat ggatgtctgg 360ggccagggca ccctggtcac
cgtctccagc 39014133PRTOryctolagus cuniculus 14Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val
Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40
45 Glu Ser Ile Ser Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser
Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Tyr Gly Thr Gln
Phe Thr Leu Thr 85 90 95 Ile Ser Gly Val Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Trp Asn Ile Asn Asn Ile Asp
Asn Ile Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Gly 130
15399DNAOryctolagus cuniculus 15atggacacca gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgcgcct atgatatgac ccagactcca
gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtgagagc attagcactt ggttagcctg gtatcagcag 180aaaccagggc
agcctcccaa gctcctgatc tacagggcat ccactctggc atctggggtc
240tcatcgcggt tcaaaggcag tggatatggg acacagttca ctctcaccat
cagcggcgtg 300gagtgtgccg atgctgccac ttactactgt caacagggtt
ggaatattaa taatattgat 360aatattttcg gcggagggac cgaggtggtg gtcaaagga
39916137PRTOryctolagus cuniculus 16Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45 Ser Tyr
Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Tyr Ile Gly Phe Ile Leu Ser Ala Thr Ala Val Ser Tyr Ala Thr Trp 65
70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val
Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Asp Arg Asp Gly Gly Thr Thr Leu Asp
Gly Phe Asp Pro Trp Gly 115 120 125 Pro Gly Thr Leu Val Thr Val Ser
Ser 130 135 17411DNAOryctolagus cuniculus 17atggagacag gcctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtagctat tcaatgtatt gggtccgcca ggctccaggg
180aaggggctgg aatacatcgg atttattctt agtgctactg ccgtatccta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccagaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag atagagatgg tggtactaca 360ctagatggtt ttgatccctg
gggcccaggc accctggtca ccgtctccag c 41118135PRTOryctolagus cuniculus
18Met Asp Ile Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Leu Leu Thr Gln Thr Pro Pro
Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys
Gln Ser Ser 35 40 45 Gln Ser Val Val Asn Asn Asn Asn Leu Ala Trp
Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr
Phe Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys
Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp
Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln Gly Thr
Tyr Leu Ser Asp Asp Trp Ser Asp Ala Phe Gly Gly Gly 115 120 125 Thr
Glu Val Val Val Lys Gly 130 135 19405DNAOryctolagus cuniculus
19atggacatca gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccc aactgctgac ccagactcca ccctcggtgt ctgcagctgt gggaggcaca
120gtcaccatca gttgccagtc cagtcagagt gttgttaata acaacaactt
agcctggtat 180cagcagaaac cagggcagcc tcccaagctc ctgatctatt
ttgcctccac tctggcatct 240ggggtcccat cgcggttcaa aggcagtgga
tctgggacac agttcactct caccatcagc 300gacgtgcagt gtgacgatgc
tgccacttac tactgtcaag gcacttatct tagtgatgat 360tggtccgatg
ctttcggcgg agggaccgag gtggtggtca aagga 40520136PRTOryctolagus
cuniculus 20Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu
Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Lys Val
Ser Gly Phe Ser Leu Ser 35 40 45 Ser Ser Val Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ala Ile Trp
Ser Gly Gly Tyr Thr Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg
Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met
Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110
Arg Gly Gln Phe Gly Ala Ser Gly Gly Gly Asp Val Leu Trp Gly Pro 115
120 125 Gly Thr Leu Val Thr Val Ser Ser 130 135 21408DNAOryctolagus
cuniculus 21atggagacag gcctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120aaagtctctg gattctccct cagttcctct gtaatgaact
gggtccgcca ggctccaggg 180aaggggctgg agtggatcgg agccatttgg
agtggtggtt acacatacta cgcgacctgg 240gcaaaaggcc gattcaccat
ctccagaacc tcgaccacgg tggatctgaa aatgaccagt 300ctgacagccg
cggacacggc cacctacttc tgtgccagag gacaatttgg tgctagtggt
360ggtggtgatg ttttgtgggg cccgggcacc ctggtcaccg tctccagc
40822136PRTOryctolagus cuniculus 22Met Asp Ile Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln
Ser Ser 35 40 45 Pro Ser Val Asp Asn Asn Asn Leu Leu Ser Trp Phe
Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp
Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Glu Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Ala Ile Ser Gly Val
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Val Ala Gly Tyr
Gly Lys Arg Ser Arg Asp Ile Arg Val Phe Gly Gly 115 120 125 Gly Thr
Gly Val Val Val Lys Gly 130 135 23408DNAOryctolagus cuniculus
23atggacataa gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca
120gtcaccatca gttgccagtc cagtccgagt gttgataata acaacctctt
atcctggttt 180cagcagaaac cagggcagcc tcccaagctc ctgatctatg
atgcttccac tctggcatct 240ggggtcccat cgcggttcga aggcagtgga
tctgggacac aattcactct cgccatcagc 300ggcgttcagt gtgacgatgc
tgccacttac tactgtgtag ccggttatgg taaaaggagt 360cgagatatac
gagttttcgg cggagggacc ggggtggtgg tcaaagga 40824130PRTOryctolagus
cuniculus 24Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu
Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val
Ser Gly Phe Asp Ile Ser 35 40 45 Ser His Asn Met Gln Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr
Pro Ser Asn Asn Ala Tyr Tyr Ser Asn Trp 65 70 75 80 Ala Lys Gly Arg
Phe Thr Ile Ser Lys Thr Ser Thr Thr Met Ser Leu 85 90 95 Gln Met
Asn Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110
Arg Asp Ile Asn Ser Ala Ile Trp Gly Pro Gly Thr Leu Val Thr Val 115
120 125 Ser Ser 130 25390DNAOryctolagus cuniculus 25atggagacag
gcctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120accgtctctg gattcgacat cagtagccac aatatgcaat gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg aatcatttat cctagtaata
atgcatacta ttccaactgg 240gcgaaaggcc gattcaccat ctccaaaact
tcgaccacga tgtctctgca aatgaacagt 300ctgacaaccg aggacacggc
cacctatttc tgcgccagag acataaatag tgctatttgg 360ggcccaggca
ccctggtcac cgtctccagc 39026134PRTOryctolagus cuniculus 26Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Val Ile Cys Ala Glu Val Ala Met Thr Gln Thr Pro Ser 20
25 30 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asp Cys Gln
Ala 35 40 45 Ser Glu Ser Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln
Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser
Asp Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Glu Tyr Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 Cys Ser Trp Gly Gly
Ser Thr Tyr Gly Phe Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val Val
Val Lys Arg 130 27402DNAOryctolagus cuniculus 27atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgtc 60atatgtgccg
aagtagcgat gacccagact ccatcctccg tgtctgcagc tgtgggaggc
120acagtcacca tcgattgcca ggccagtgag agcatttata gcaatttagc
ctggtatcag 180cagaaaccag ggcagcctcc caagctcctg atctatgatg
catccgatct ggcatctggg 240gtcccatcgc ggttcaaagg cagtggatct
gggacagagt acactctcac catcagcgac 300ctggagtgtg ccgatgctgc
cacttactac tgtcaatgta gttggggtgg tagtacttat 360ggttttgctt
tcggcggagg gaccgaggtg gtggtcaaac gt 40228135PRTOryctolagus
cuniculus 28Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu
Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val
Ser Gly Ile Asp Leu Ser 35 40 45 Ile Tyr Val Met Thr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ser Ile Asp
Ala Asp Asp Ser Ala Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Thr Ser Arg
Ser Thr Ile Ser Arg Thr Ser Thr Thr Val Ala Leu 85 90 95 Ser Ile
Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110
Arg Gly Leu Tyr Ala Asn Gly Gly Pro Phe Thr Leu Trp Gly Pro Gly 115
120 125 Thr Leu Val Thr Val Ser Ser 130 135 29405DNAOryctolagus
cuniculus 29atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggta acgcctggga cacccctgac
actcacctgc 120acagtctctg gaatcgacct cagtatctat gtaatgactt
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aagcatcgat
gctgacgata gcgcatacta cgcgacctgg 240gcgacaagcc gatccaccat
ctccagaacc tcgaccacgg tggctctgag catcaccagt 300ccgacaaccg
aggacacggc gacctatttc tgtgccaggg gactatatgc taatggtggt
360ccctttacct tatggggccc aggcaccctc gtcaccgtct cgagc
40530135PRTOryctolagus cuniculus 30Met Asp Met Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser 35 40 45 Pro Ser
Val Tyr Asn Arg Asn Gln Leu Ser Trp Phe Gln Gln Lys Ala 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Phe Thr Ala Ser Thr Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Gln Gly Tyr Tyr Asn Thr Gly Ser Asp Thr
Tyr Ala Phe Gly Gly Gly 115 120 125 Thr Glu Val Val Val Lys Gly 130
135 31405DNAOryctolagus cuniculus 31atggacatga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccg ccgtgctgac
ccagactcca tctcccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
agtgccagtc cagtccgagt gtttataatc gcaaccaatt atcctggttt
180cagcagaaag cagggcagcc tcccaagctc ctgatcttta ctgcgtccac
tctggcatct 240ggggtcccat cgcggttcaa aggcagtgga tctgggacag
atttcactct caccatcagc 300gacctggagt gtgccgatgc tgccacttac
tactgtcaag gctattataa tactggtagt 360gatacgtatg ctttcggcgg
agggaccgag gtggtggtca aagga 40532133PRTOryctolagus cuniculus 32Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Asn 35 40 45 Asn Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Tyr Ile Gly Phe Ile Asn Thr Val Gly Tyr
Ala Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Arg Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Ser Leu Asp Asn
Tyr Tyr Thr Trp Gly Ile Trp Gly Pro Gly Thr Leu 115 120 125 Val Thr
Val Ser Leu 130 33399DNAOryctolagus cuniculus 33atggagacag
gcctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120acagtctctg gattctccct caataactac gacatgagct gggtccgcca
ggctccaggg 180aaggggctgg aatatatcgg attcattaat actgttggtt
acgcatacta cgcgagctgg 240gcaaaaggcc gattcaccat ctccagaacc
tcgaccacgg tggatctgaa aatgaccagt 300ctgacaaccg aggacacggc
cacctatttc tgtgccagcc ttgataatta ctatacttgg 360ggcatctggg
gcccaggcac cctggtcacc gtttccata 39934134PRTOryctolagus cuniculus
34Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Pro Gly Ala Arg Cys Ala Asp Ile Val Met Thr Gln Thr Pro
Gly 20 25 30 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn
Cys Gln Ala 35 40 45 Ala Glu Asp Ile Tyr Ser Ser Leu Ala Trp Tyr
Gln Gln Lys Ser Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Ala
Ala Ser Ile Leu Ala Ser Gly 65 70 75 80 Ala Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Glu Tyr Thr Leu 85 90 95 Thr Ile Ser Gly Val
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 Thr Asn Tyr
Gly Ile Ser Ser Tyr Gly Ala Ala Phe Gly Gly Gly Thr 115 120 125 Glu
Val Val Val Lys Gly 130 35402DNAOryctolagus cuniculus 35atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgccg
acatcgtgat gacccagact ccaggctccg tgtctgcagc tgtgggaggc
120acagtcacca tcaattgcca ggccgctgag gacatttata gctctttggc
ctggtatcag 180cagaaatcag ggcagcctcc caagctcctg atctatgctg
catctattct ggcatctggg 240gccccatcgc ggttcagtgg cagtggatct
gggacagagt acactctcac catcagcggc 300gtgcagtgtg acgatgctgc
cacttactac tgtcaaacca attatggtat cagtagttat 360ggtgcggctt
tcggcggagg gaccgaggtg gtggtcaaag ga 40236134PRTOryctolagus
cuniculus 36Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu
Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Ser 35 40 45 Asn Lys Pro Ile Thr Trp Val Arg
Gln Ala Pro Gly Glu Gly Leu Glu 50 55 60 Tyr Ile Gly Trp Ile Ser
Thr Thr Gly Ser Ala Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg
Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Ile
Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110
Arg Tyr Ser Ser Asp Tyr Gly His His Asp Leu Trp Gly Pro Gly Thr 115
120 125 Leu Val Thr Val Ser Ser 130 37402DNAOryctolagus cuniculus
37atggagacag gcctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag
60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120accgtctctg ggttctccct cagtaacaag ccaataacat gggtccgcca
ggctccaggg 180gaggggctgg aatacatcgg atggattagt actactggta
gcgcatacta cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc
cacctatttc tgtgccagat atagtagtga ttatggacat 360catgacttgt
ggggcccagg caccctggtc accgtctcca gc 40238132PRTOryctolagus
cuniculus 38Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu
Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln
Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala Leu Gly Gly Ser Val Thr
Ile Asn Cys Gln Ala Ser 35 40 45 Gln Ser Val Tyr Arg Asp Tyr Leu
Ser Trp Phe Gln Gln Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu
Ile Asn Gly Ala Ser Asn Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr Ile
Ser Asp Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu 100 105 110
Gly Gly Phe Ser Gly Asn Ile Asn Thr Phe Gly Gly Gly Thr Glu Val 115
120 125 Val Val Lys Gly 130 39396DNAOryctolagus cuniculus
39atggacacca gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgcgc aagtgctgac ccagactcca tcacccgtgt ctgcagctct gggaggctca
120gtcaccatca attgccaggc cagtcagagt gtttatagag attacttatc
ctggtttcag 180cagaaaccag gtcagcctcc caagctcctg atcaatggtg
catccaatct ggcatctggg 240gtcccatcgc ggttcagcgg cagtggatct
gggacacagt tcactctcac catcagcgac 300gtgcagtgtg acgatgctgc
cacttactac tgtctaggcg gttttagtgg taatatcaat 360actttcggcg
gagggaccga ggtggtggtc aaggga 39640131PRTOryctolagus cuniculus 40Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp
Leu Asn 35 40 45 Asn Tyr His Met Cys Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Lys 50 55 60 Tyr Ile Gly Ile Ile Ser Asn Thr Gly Tyr
Thr Tyr Tyr Ser Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Gly Asp Arg
Leu Ala Asn Leu Trp Gly Pro Gly Thr Leu Val Thr 115 120 125 Val Ser
Ser 130 41393DNAOryctolagus cuniculus 41atggagacag gcctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggta acgcctggag gatccctgac actcacctgt 120acagtctctg
gaatcgacct caataactac cacatgtgct gggtccgcca ggctccaggg
180aaggggctga aatacatcgg aatcattagt aatactggtt acacatacta
ctcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca
cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgtgctg gagaccggct tgctaacttg 360tggggcccag gcaccctggt
cactgtctcc agc 39342136PRTOryctolagus cuniculus 42Met Asp Thr Arg
Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro
Gly Ala Arg Cys Ala Asp Val Val Met Thr Gln Thr Pro Ala 20 25 30
Ser Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 35
40 45 Ser Gln Ser Ile Ser Thr Tyr Leu Lys Trp Tyr Gln Gln Lys Pro
Gly 50 55 60 Gln Arg Pro Lys Arg Leu Ile Tyr Ser Ala Ser Thr Leu
Thr Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Gly Asp
Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 Ser Asn Ala Gly Ser Ser Ser
Ser Ser Cys Gly Tyr Ala Phe Gly Gly 115 120 125 Gly Thr Glu Val Val
Val Lys Gly 130 135 43408DNAOryctolagus cuniculus 43atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgccg
acgtcgtgat gacccagact ccagcctccg tggaggcagc tgtgggaggc
120acagtcacca tcaagtgcca ggccagtcag agcattagta cctacttaaa
gtggtatcag 180cagaaaccag gacagcgtcc caagcgcctg atctattctg
catccactct gacatctggg 240gtcccatcgc ggttcaaagg cagtggatct
gggacagatt tcactctcac catcagcgac 300ctggagtgtg gcgatgctgc
cacttactac tgtcaaagca atgctggtag tagtagtagt 360agttgtggtt
atgctttcgg cggagggacc gaggtggtgg tcaaagga 40844127PRTOryctolagus
cuniculus 44Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu
Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu
Ser 35 40 45 Tyr Tyr Gly Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Tyr Ile Gly Ile Ile Ser Gly Ile Gly Asn Thr
Tyr Tyr Pro Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Pro Thr Thr Val Asp Leu 85 90 95 Arg Ile Thr Ser Pro Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Thr Gly Asp Phe Trp
Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 125
45381DNAOryctolagus cuniculus 45atggagacag gcctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggga cacccctgac actcacctgc 120acagtctctg gaattgacct
cagttactat gggatgggct gggtccgcca ggctccaggg 180aaggggctgg
aatacatcgg gatcattagt ggtattggta atacatacta tccgacctgg
240gcgaaaggcc gattcaccat ctccaaaacc ccgaccacgg tggatctaag
gatcaccagt 300ccgacaaccg aggacacggc cacctatttc tgtgccactg
gggacttctg gggcccaggc 360accctggtca ccgtctccag c
38146136PRTOryctolagus cuniculus 46Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Asp Val Val Met Thr Gln Thr Pro Ala 20 25 30 Ser Val Ser Glu
Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 35 40 45 Ser Gln
Ser Ile Ser Arg Tyr Leu Lys Trp Tyr Gln Gln Lys Pro Gly 50 55 60
Gln Arg Pro Lys Arg Leu Ile Tyr Ser Ala Ser Thr Leu Thr Ser Gly 65
70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr
Tyr Tyr Cys Gln 100 105 110 Ser Asn Ala Gly Ser Ser Ser Ser Ser Cys
Gly Tyr Ala Phe Gly Gly 115 120 125 Gly Thr Glu Val Val Val Lys Gly
130 135 47408DNAOryctolagus cuniculus 47atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgccg acgtcgtgat
gacccagact ccagcctccg tgtctgaacc tgtgggaggc 120acagtcacca
tcaagtgcca ggccagtcag agcattagta ggtacttaaa gtggtatcag
180cagaaaccag ggcagcgtcc caagcgcctg atctattctg catccactct
gacatctggg 240gtcccatcgc ggttcaaagg cagtggatct gggacagatt
tcactctcac catcagcgac 300ctggagtgtg ccgatgctgc cacttactac
tgtcaaagca atgctggtag tagcagtagt 360agttgtggtt atgctttcgg
cggagggacc gaggtggtgg tcaaagga 40848127PRTOryctolagus cuniculus
48Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Val
Asp Leu Ser 35 40 45 Tyr Tyr Gly Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Tyr Ile Gly Ile Ile Ser Gly Ser Gly
Asn Thr Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Ser Gly Asp
Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 125
49381DNAOryctolagus cuniculus 49atggagacag gcctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggga cacccctgac actcacctgc 120acagtctctg gagtcgacct
cagttactat ggaatgggct gggtccgcca ggctccaggg 180aaggggctgg
aatacatcgg gatcattagt ggtagtggta acacatacta cgcgagctgg
240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgaa
aatcaccagt 300ccgacaaccg aggacacggc cacctatttc tgtgccagtg
gggacttctg gggcccaggc 360accctggtca ccgtctccag c
38150134PRTOryctolagus cuniculus 50Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Leu Val Met Thr Gln Thr Ala Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Ser Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly 50 55 60
Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Ser Leu Ala Ser Gly 65
70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe
Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr
Tyr Tyr Cys Gly 100 105 110 Gly Phe Gln Lys Tyr Ile Asp Asp Gly Gly
Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val Val Val Lys Gly 130
51402DNAOryctolagus cuniculus 51atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgccc ttgtgatgac ccagactgca
tcccccgtgt ctgcagctgt gggaggcaca 120gtcaccatca gttgccagtc
cagtcagagt gtttatagta actacttatc ctggtttcag 180cagaaaccag
ggcagcctcc caagctcctg atctatgttg catccagtct ggcatctggg
240gtcccatcgc gattcaaagg cagtggatct gggacacagt tcactctcac
catcagtgac 300ctggagtgtg acgatgctgc cacttactac tgtggaggct
ttcagaaata tattgatgat 360gggggggctt tcggcggagg gaccgaggtg
gtggtcaaag ga 40252131PRTOryctolagus cuniculus 52Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Thr Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 50 55 60 Trp Ile Gly Ile Met Ser Ser Ser Gly Ser Ala Tyr Tyr
Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ala
Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Ile Glu
Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Ser Ser Ser Phe Gly
Leu Trp Gly Pro Gly Thr Leu Val Thr 115 120 125 Val Ser Ser 130
53393DNAOryctolagus cuniculus 53atggagacag gcctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggag gatccctgac acttacctgc 120acagtctctg gaatcgacct
cagtacctat gcaatgagtt gggtccgcca ggctccaggg 180aaggggctgg
aatggatcgg aatcatgagt agtagtggta gcgcatacta cgcgagctgg
240gcgaaaggcc gattcaccat ctccaaaacc gcgtcgacca cggtggatct
gaaaatgacc 300agtctgacaa tcgaggacac ggccacctat ttctgtgcca
gaagttctag ttttggattg 360tggggcccag gcaccctggt caccgtctcc agc
39354106PRTMus musculus 54Thr Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro Ser Ser Glu Gln 1 5 10 15 Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe Tyr 20 25 30 Pro Lys Asp Ile Asn Val
Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 35 40 45 Asn Gly Val Leu
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser
Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 85
90 95 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 55324PRTMus
musculus 55Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu
Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn
Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val
Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 65 70 75 80 Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val
Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115
120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile
Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln Pro Arg Glu
Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro
Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys
Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr
Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235
240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr
Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His
His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys
56130PRTMus musculus 56Met Asp Phe Gln Val Gln Ile Phe Ser Phe Met
Leu Ile Ser Val Thr 1 5 10 15 Val Ile Leu Ser Ser Gly Glu Ile Val
Leu Thr Gln Ser Pro Ala Leu 20 25 30 Met Ala Ala Ser Pro Gly Glu
Lys Val Thr Ile Ala Cys Ser Val Ser 35 40 45 Ser Ser Ile Ser Ser
Thr Asn Leu His Trp Ser Gln Gln Lys Ser Gly 50 55 60 Thr Ser Pro
Lys Leu Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly 65 70 75 80 Val
Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 85 90
95 Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
100 105 110 Gln Trp Ser Thr Thr Tyr Thr Phe Gly Ser Gly Thr Lys Leu
Glu Leu 115 120 125 Lys Arg 130 57387DNAMus musculus 57atggattttc
aagtgcagat tttcagcttc atgctaatca gtgtcacagt catattgtcc 60agtggagaaa
ttgtgctcac ccagtctcca gcactcatgg ctgcatctcc aggggagaag
120gtcaccatcg cctgcagtgt cagctcaagt ataagttcca ccaacttaca
ctggtcccag 180cagaagtcag gaacctcccc caaactctgg atttatggca
catccaacct tgcttctgga 240gtccctgttc gcttcagtgg cagtggatct
gggacctctt attctctcac aatcagcagc 300atggaggctg aagatgctgc
cacctattac tgtcaacagt ggagtactac gtatacgttc 360ggatcgggga
ccaagctgga gctgaaa 38758141PRTMus musculus 58Met Gly Trp Asn Trp
Ile Ile Phe Phe Leu Met Ala Val Val Thr Gly 1 5 10 15 Val Asn Ser
Glu Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Val Lys 20 25 30 Pro
Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40
45 Lys Asp Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Ala Gln Gly Leu
50 55 60 Glu Trp Ile Gly Arg Ile Asp Pro Asp Asn Gly Glu Ser Thr
Tyr Val 65 70 75 80 Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp
Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Leu Gln Leu Arg Ser Leu Thr
Ser Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Gly Arg Glu Gly Leu
Asp Tyr Gly Asp Tyr Tyr Ala Val 115 120 125 Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser 130 135 140 59420DNAMus musculus
59atgggatgga actggatcat cttcttcctg atggcagtgg ttacaggggt caattcagag
60gtgcagttgc agcagtctgg ggcagacctt gtgaagccag gggcctcagt caagttgtcc
120tgcacagctt ctggcttcaa cattaaagac tactatatac actgggtgaa
gcagaggcct 180gcacagggcc tggagtggat tggaaggatt gatcctgata
atggtgaaag tacatatgtc 240ccgaagttcc agggcaaggc cactataaca
gcagacacat catccaacac agcctaccta 300caactcagaa gcctgacatc
tgaggacact gccatctatt attgtgggag agaggggctc 360gactatggtg
actactatgc tgtggactac tggggtcaag gaacctcagt cactgtctcg
42060132PRTMus musculus 60Met Glu Ser Asp Thr Leu Leu Leu Trp Val
Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Lys Ile Val Leu
Thr Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Arg Gln Arg
Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser 35 40 45 Val Asp Ser Tyr
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln
Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser 65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr 85
90 95 Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr
Cys 100 105 110 Gln Gln Asn Tyr Glu Asp Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu 115 120 125 Glu Leu Lys Arg 130 61393DNAMus musculus
61atggagtcag acacactcct gctatgggtg ctactgctct gggttccagg ttccacaggt
60aaaattgtac tgacccaatc tccagcttct ttggctgtgt ctctaaggca gagggccacc
120atatcctgca gagccagtga aagtgttgat agttatggca atagttttat
gcactggtac 180cagcagaaac caggacagcc acccaaactc ctcatctatc
gtgcatccaa cctagaatct 240ggggtccctg ccaggttcag tggcagtggg
tctaggacag acttcaccct caccattgat 300cctgtggagg ctgatgatgc
tgcaacctat tactgtcagc aaaattatga ggatccgctc 360acgttcggtg
ctgggaccaa gttggagctg aaa 39362145PRTMus musculus 62Met Gly Trp Pro
Trp Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His
Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35
40 45 Thr Asn Tyr Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly
Leu 50 55 60 Glu Trp Ile Gly Val Ile Asn Pro Gly Ser Gly Ile Ile
Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ile Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Asp Trp
Asp Thr Phe Tyr Ser Tyr Glu Arg Glu 115 120 125 Val Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 130 135 140 Ser 145
63432DNAMus musculus 63atgggatggc cctgggtctt tatatttctc ctgtcagtaa
ctgcaggtgt tcactcccag 60gtccagctgc agcagtctgg agctgagctg gtaaggcctg
ggacctcagt gaaggtgtcc 120tgcaaggcct ctggatacgc cttcactaat
tacttgatag agtggataaa gcagaggcct 180ggacagggcc ttgagtggat
tggagtgatt aatcctggaa gcggtattat taattacaat 240gagaagttca
aaatcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg
300cagctcagca gcctgacatc tgatgattct gcggtctatt tctgtgcaag
agactgggat 360actttctata gttacgagag ggaggtctat gctatggact
actggggtca agggacctca 420gtcaccgtct cg 43264128PRTMus musculus
64Met Glu Ser Gln Thr Leu Val Leu Val Phe Leu Leu Phe Trp Ile Pro 1
5 10 15 Ala Ser Arg Gly Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu
Ser 20 25 30 Val Ser Pro
Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile
Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asp Gly Ser Pro 50 55
60 Arg Leu Leu Ile Lys Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser
65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser
Ile Asn 85 90 95 Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys
Gln Gln Ser Asn 100 105 110 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys Arg 115 120 125 65381DNAMus musculus
65atggaatcac agactctggt ccttgtattt ttgcttttct ggattccagc ctccagaggt
60gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt
120ttctcctgca gggccagtca gagcattggc acaaacatac actggtatca
gcaaagaaca 180gatggttctc caaggcttct cataaagttt gcttctgagt
ctatctctgg gatcccttcc 240aggtttagtg gcagtggctc agggacagat
tttactctta gcatcaacag tgtggagtct 300gaagatattg cagattatta
ctgtcaacaa agtaatagct ggccactcac gttcggtgct 360gggaccaagc
tggaactgaa a 38166137PRTMus musculus 66Met Asp Trp Ser Trp Ile Ile
Phe Phe Leu Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Ser Gly Ala
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Glu
Asp Tyr Tyr Val His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55
60 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala
65 70 75 80 Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser
Ser Asn 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Gly Gly Tyr Gly Asn Phe
Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser
Ser 130 135 67408DNAMus musculus 67atggattgga gctggatcat cttcttcctg
atggcagtgg ttataggaat caattcagag 60gttcagctgc agcagtctgg ggcagagctt
gtgaggtcag gggcctcagt caagttgtcc 120tgcacagctt ctggcttcaa
cattgaagac tactatgtgc actgggtgaa gcagaggcct 180gaacagggcc
tggagtggat tggatggatt gatcctgaga atggtgatac tgaatatgcc
240ccgaagttcc agggcaaggc cactatgact gcagacacat cctccaacac
agcctacctg 300cagctcagca gcctgacatc tgaggacact gccgtctatt
actgtaatgg ggggtatggt 360aacttttact ttgactactg gggccaaggc
accactgtca ccgtctcg 40868128PRTMus musculus 68Met Glu Phe Gln Thr
Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser 1 5 10 15 Gly Val Glu
Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser 20 25 30 Thr
Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35 40
45 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
50 55 60 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val
Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser 85 90 95 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr
Phe Cys Gln Gln Tyr Ser 100 105 110 Ser Tyr Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Leu Lys Arg 115 120 125 69381DNAMus musculus
69atggaattcc agactcaggt ctttgtatac atgttgctgt ggttgtctgg tgttgaagga
60gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc
120atcacctgca aggccagtca ggatgtgggt actgctgtag cctggtatca
acagaaacca 180gggcaatctc ctaaactact gatttactgg gcatccaccc
ggcacactgg agtccctgat 240cgcttcacag gcagtggatc tgggacagat
ttcactctca ccattagcaa tgtgcagtct 300gaagacttgg cagattattt
ctgtcagcaa tatagcagct atccgtacac gttcggaggg 360gggaccaagc
tggagctgaa a 38170137PRTMus musculus 70Met Lys Cys Asn Trp Val Ile
Phe Phe Leu Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Ser Gly Ala
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys
Asp Tyr Phe Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55
60 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala
65 70 75 80 Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser
Ser Asn 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala Gly Gln Ala Thr Tyr
Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser
Ser 130 135 71408DNAMus musculus 71atgaaatgca actgggtcat cttcttcctg
atggcagtgg ttataggaat caattcagag 60gttcagctgc agcagtctgg ggcagagctt
gtgaggtcag gggcctcagt caagttgtcc 120tgcacagctt ctggcttcaa
cattaaagac tactttatgc actgggtgaa gcagaggcct 180gaacagggcc
tggagtggat tggatggatt gatcctgaga atggtgatac tgaatatgcc
240ccgaagttcc agggcaaggc cactatgact gcagacacat cctccaacac
agcctacctg 300cagctcagca gcctgacatc tgaggacact gccgtctatt
actgtaatgc agggcaggca 360acttactact ttgactactg gggccaaggc
accacggtca ccgtctcg 40872133PRTMus musculus 72Met Glu Thr Asp Thr
Leu Arg Ala Lys Leu Arg Ser His His Glu Ala 1 5 10 15 Ala Cys Ser
Ala Phe Arg Cys Glu Met Asp Ile Gln Met Asn Gln Ser 20 25 30 Pro
Ser Ser Leu Ser Ala Ser Leu Gly Asp Thr Ile Thr Ile Thr Cys 35 40
45 His Ala Thr Gln Asn Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys
50 55 60 Pro Gly Asn Ile Pro Thr Leu Leu Ile Tyr Lys Thr Ser Asn
Leu His 65 70 75 80 Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Gly Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu
Asp Ile Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Gly Gln Ser Phe Pro
Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Leu Lys Arg 130
73397DNAMus musculus 73atggagacag acacactccg agcgaagctt cgaagccacc
atgaagctgc ctgttctgct 60tttaggtgtg agatgtgaca tccagatgaa ccagtctcca
tccagtctgt ctgcatccct 120tggagacaca attaccatca cttgccatgc
cactcagaac attaatgttt ggttaagctg 180gtaccagcag aaaccaggaa
atattcctac acttttgatc tataagactt ccaacttgca 240cacaggcgtc
ccatcaaggt ttagtggcag tggatctgga acaggtttca cattaaccat
300cagcagcctg cagtctgaag acattgccac ttactactgt caacagggtc
aaagttttcc 360gtacacgttc ggaggaggca ccaagctgga gctgaaa
39774137PRTMus musculus 74Met Lys Cys Asn Trp Val Ile Phe Phe Leu
Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser Glu Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Ser Gly Ala Ser Val Lys
Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Phe
Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp
Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala 65 70 75 80
Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn 85
90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val 100 105 110 Tyr Tyr Cys Asn Ala Gly Gln Ala Thr Tyr Tyr Phe Asp
Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser 130 135
75408DNAMus musculus 75atgaaatgca actgggtcat cttcttcctg atggcagtgg
ttataggaat caattcagag 60gttcagctgc agcagtctgg ggcagagctt gtgaggtcag
gggcctcagt caagttgtcc 120tgcacagctt ctggcttcaa cattaaagac
tactttatgc actgggtgaa gcagaggcct 180gaacagggcc tggagtggat
tggatggatt gatcctgaga atggtgatac tgaatatgcc 240ccgaagttcc
agggcaaggc cactatgact gcagacacat cctccaacac agcctacctg
300cagctcagca gcctgacatc tgaggacact gccgtctatt actgtaatgc
agggcaggca 360acttactact ttgactactg gggccaaggc accacggtca ccgtctcg
40876134PRTMus musculus 76Met Glu Ser Gln Thr Gln Val Leu Met Leu
Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Met Met
Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys
Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg 65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85
90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val
Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Asp Tyr Pro Trp Thr Phe Gly
Gly Gly Ser 115 120 125 Lys Leu Glu Leu Lys Arg 130 77399DNAMus
musculus 77atggaatcac agacccaggt tcttatgtta ctgctgctat gggtatctgg
tacctgtggg 60gacattatga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga
gaaggttact 120atgagctgca agtccagtca gagcctttta tatagtaaca
atcaaaagaa ctacttggcc 180tggtaccagc agaaaccagg gcagtctcct
aaactgctga tttcctgggc atccactagg 240gaatctgggg tccctgatcg
cttcacaggc agtggatctg ggacagattt cactctcacc 300atcagcagtg
tgaaggctga agacctggct gtttattact gtcagcaata ttatgactat
360ccgtggacgt tcggtggagg ctccaagctg gagctgaaa 39978137PRTMus
musculus 78Met Gly Trp Asn Trp Ile Ile Phe Phe Leu Met Ala Val Val
Ile Gly 1 5 10 15 Ile Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg 20 25 30 Ser Gly Ala Ser Val Lys Leu Ser Cys Thr
Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Phe Met His Trp Val
Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile
Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala 65 70 75 80 Pro Lys Phe Gln
Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala
Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110
Tyr Tyr Cys Asn Ala Gly Gln Ala Thr Tyr Tyr Phe Asp Tyr Trp Gly 115
120 125 Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 79408DNAMus
musculus 79atgggatgga actggatcat cttcttcctg atggcagtgg ttataggaat
caattcagag 60gttcagctgc agcagtctgg ggcagagctt gtgaggtcag gggcctcagt
caagttgtcc 120tgcacagctt ctggcttcaa cattaaagac tactttatgc
actgggtgaa gcagaggcct 180gaacagggcc tggagtggat tggatggatt
gatcctgaga atggtgatac tgaatatgcc 240ccgaagttcc agggcaaggc
cactatgact gcagacacat cctccaacac agcctacctg 300cagctcagca
gcctgacatc tgaggacact gccgtctatt actgtaatgc agggcaggca
360acttactact ttgactactg gggccaaggc accacggtca ccgtctcg
40880130PRTMus musculus 80Met Asp Met Arg Thr Pro Ala Gln Phe Leu
Gly Leu Leu Leu Leu Cys 1 5 10 15 Phe Gln Gly Thr Arg Cys Asp Ile
Gln Met Thr Gln Thr Ser Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly
Asp Arg Val Thr Ile Ser Cys Arg Thr Ser 35 40 45 Gln Asp Ile Asn
Asn Phe Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val
Lys Phe Leu Ile His His Thr Ser Arg Leu Lys Ser Gly Val 65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85
90 95 Ile Ser His Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
His 100 105 110 Tyr Tyr Asn Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Leu 115 120 125 Lys Arg 130 81387DNAMus musculus
81atggacatga ggacccctgc tcagttcctt ggtctcctgt tgctctgttt tcaaggtacc
60agatgtgata tccagatgac acagacttca tcttccctgt ctgcctctct gggagacaga
120gtcaccatca gttgcaggac aagtcaggat attaacaatt ttttaaactg
gtatcagcag 180aaaccagatg gaactgttaa attcctgatc caccacacat
caagattaaa gtcaggagtc 240ccatcaaggt tcagtggcag tgggtctggg
acagattatt ctctcaccat cagccacctg 300gaacctgaag atattgccac
ttactattgt cagcactatt ataatcttcc gtggacgttc 360ggtggaggca
ccaagttgga gctgaaa 38782138PRTMus musculus 82Met Gly Trp Ser Trp
Thr Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser
Glu Ile Gln Leu Gln Gln Thr Gly Pro Glu Leu Val Lys 20 25 30 Pro
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40
45 Thr Asp Asn Ile Met Val Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60 Glu Trp Ile Gly Asn Ile Asn Pro Tyr Tyr Gly Ser Pro Ser
Tyr Asn 65 70 75 80 Leu Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr
Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Gly Gly
Asn Tyr Gly Ser Leu Asp Asn Trp 115 120 125 Gly Gln Gly Thr Ser Val
Thr Val Ser Ser 130 135 83411DNAMus musculus 83atgggatgga
gctggacctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactctgag 60atccagctgc
agcagactgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc
120tgcaaggctt ctggttattc attcactgac aacatcatgg tctgggtgaa
gcagagccat 180ggaaagagcc ttgagtggat tggaaacatt aatccttact
atggtagtcc tagttacaat 240ctgaagttca aggacaaggc cacattgact
gtagacaaat cttccagcac agcctacatg 300cagctcaata gtctgacatc
tgaggactct gcagtctatt actgtgcaag agaggggggt 360aattacggtt
ctctggacaa ctggggtcaa ggaacctcgg tcaccgtctc g 41184134PRTMus
musculus 84Met Lys Ser Gln Thr Gln Val Leu Met Leu Leu Leu Leu Trp
Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Met Met Ser Gln Ser Pro
Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser
Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Asn Asn Gln Lys
Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr
Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110
Tyr Cys Gln Gln Tyr Tyr Asp Tyr Pro Trp Thr Phe Gly Gly Gly Ser 115
120 125 Lys Leu Glu Ile Lys Arg 130 85399DNAMus musculus
85atgaagtcac agacccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg
60gacattatga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact
120atgagctgca agtccagtca gagcctttta tatagtaaca atcaaaagaa
ctacttggcc 180tggtaccagc agaaaccagg gcagtctcct aaactgctga
tttcctgggc atccactagg 240gaatctgggg tccctgatcg cttcacaggc
agtggatctg ggacagattt cactctcacc 300atcagcagtg tgaaggctga
agacctggct gtttattact gtcagcaata ttatgactat 360ccgtggacgt
tcggtggagg ctccaagctg gaaataaaa 39986138PRTMus musculus 86Met Gly
Trp Ser Trp Thr Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15
Val His Ser Glu Ile Gln Leu Gln Gln Thr Gly Pro Glu Leu Val Lys 20
25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe 35 40 45 Thr Asp Asn Ile Met Val Trp Val Lys Gln Ser His Gly
Lys Ser Leu 50 55 60 Glu Trp Ile Gly Asn Ile Asn Pro Tyr Tyr Gly
Ser Pro Ser Tyr Asn 65 70 75 80 Leu Lys Phe Lys Asp Lys Ala Thr Leu
Thr Val
Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Gly
Gly Asn Tyr Gly Ser Leu Asp Asn Trp 115 120 125 Gly Gln Gly Thr Ser
Val Thr Val Ser Ser 130 135 87411DNAMus musculus 87atgggatgga
gctggacctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactctgag 60atccagctgc
agcagactgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc
120tgcaaggctt ctggttattc attcactgac aacatcatgg tctgggtgaa
gcagagccat 180ggaaagagcc ttgagtggat tggaaacatt aatccttact
atggtagtcc tagttacaat 240ctgaagttca aggacaaggc cacattgact
gtagacaaat cttccagcac agcctacatg 300cagctcaata gtctgacatc
tgaggactct gcagtctatt actgtgcaag agaggggggt 360aattacggtt
ctctggacaa ctggggtcaa ggaacctcgg tcaccgtctc g 41188127PRTMus
musculus 88Met Ser Pro Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe
Gln Gly 1 5 10 15 Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Ser Ser
Ser Leu Ser Ala 20 25 30 Ser Leu Gly Asp Arg Val Thr Ile Ser Cys
Arg Thr Ser Gln Asp Ile 35 40 45 Asn Asn Phe Leu Asn Trp Tyr Gln
Gln Lys Pro Asp Gly Thr Val Lys 50 55 60 Phe Leu Ile His His Thr
Ser Arg Leu Lys Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser His 85 90 95 Leu Glu
Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Tyr Asn 100 105 110
Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120
125 89378DNAMus musculus 89atgagtcctg cccagttcct tggtctcctg
ttgctctgtt ttcaaggtac cagatgtgat 60atccagatga cacagacttc atcttccctg
tctgcctctc tgggagacag agtcaccatc 120agttgcagga caagtcagga
tattaacaat tttttaaact ggtatcagca gaaaccagat 180ggaactgtta
aattcctgat ccaccacaca tcaagattaa agtcaggagt cccatcaagg
240ttcagtggca gtgggtctgg gacagattat tctctcacca tcagccacct
ggaacctgaa 300gatatcgcca cttactattg tcagcactat tataatcttc
cgtggacgtt cggtggaggc 360acaaagttgg aaataaaa 37890134PRTMus
musculus 90Met Gly Trp Ser Trp Thr Phe Leu Phe Leu Leu Ser Gly Thr
Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala
Val Ser Gly Phe Ser Leu 35 40 45 Ser Tyr Tyr Tyr Met Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Ile Gly Thr Ile
Ser Ile Asp Gly Ser Thr Tyr Tyr Ala Ser 65 70 75 80 Trp Ala Glu Gly
Arg Phe Thr Ile Ser Lys Asp Ser Thr Thr Val Tyr 85 90 95 Leu Gln
Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 100 105 110
Ala Arg Gly His Ile Asn Thr Gly Met Asp Pro Trp Gly Gln Gly Thr 115
120 125 Pro Val Thr Val Ser Ser 130 91399DNAMus musculus
91atgggatgga gctggacctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactctgag
60gttcagctcg ttgaatccgg aggcggactc gtgaagcccg ggggcagtct taaactgtcc
120tgcgccgtca gcgggttttc cctcagttac tattacatgt catgggtgcg
gcagaccccc 180gagaaaaggt tggaatggat tggcaccatc tcaattgacg
gaagcacata ttacgcatct 240tgggccgagg gtcgcttcac aatttctaag
gacagtacta ccgtgtatct gcagatgtcc 300agccttaaga gcgaagatac
agccatgtac tactgtgcta gagggcacat caacactgga 360atggatcctt
ggggccaggg caccccggtc acagtctcg 39992132PRTMus musculus 92Met Lys
Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15
Ser Ser Ser Asp Val Leu Met Thr Gln Asn Pro Leu Ser Leu Pro Val 20
25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr
Ile 35 40 45 Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Glu Val
Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His
Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Leu Lys
Arg 130 93393DNAMus musculus 93atgaagttgc ctgttaggct gttggtgctg
atgttctgga ttcctgcttc cagcagtgat 60gttttgatga cccaaaatcc actctccctg
cctgtcagtc ttggagatca agcctccatc 120tcctgcagat caagtcagac
cattgtacat agtaatggaa acacctattt agaatggtac 180ctgcagaaac
caggccagtc tccaaagctc ctgatctacg aagtttccaa ccgattttct
240ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact
caaaatcagc 300agagtggagg ctgaggatct gggaatttat tactgctttc
aaggttcaca ttttccgcac 360acgttcggag gggggaccaa gctggaactg aaa
39394143PRTMus musculus 94Met Glu Trp Ser Gly Val Ile Phe Phe Leu
Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser Glu Val Gln Leu Gln
Gln Ser Gly Ala Glu Phe Val Arg 20 25 30 Ser Gly Ala Ser Val Lys
Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Tyr
Met Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp
Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Cys Ala 65 70 75 80
Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn 85
90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val 100 105 110 Tyr Tyr Cys Asn Ala Asp Arg Tyr Asp Glu Gly Ala Ala
Ser Asp Tyr 115 120 125 Gly Met Asp Tyr Trp Gly Gln Gly Ser Ser Val
Thr Val Ser Ser 130 135 140 95426DNAMus musculus 95atggaatgga
gcggggtcat cttcttcctg atggcagtgg ttataggaat caattcagag 60gttcagctgc
agcagtctgg ggcagagttt gtgaggtcag gggcctcagt caagttgtcc
120tgcacagctt ctggcttcaa cattaaagac tactatatgt attgggtgaa
acagaggcct 180gaacagggcc tggagtggat tggatggatt gatcctgaga
atggtgatac tgaatgtgcc 240ccgaagttcc agggcaaggc cactatgact
gcagacacat cctccaacac agcctacctg 300caactcagca gcctgacatc
tgaggacact gccgtctatt actgtaatgc cgataggtac 360gacgaggggg
ccgcgtcgga ctatggtatg gactactggg gtcaaggaag ttcagtcaca 420gtctcg
42696132PRTMus musculus 96Met Lys Leu Pro Val Arg Leu Leu Val Leu
Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser Asp Val Leu Met Thr
Gln Asn Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35 40 45 Val His Ser Asn
Gly Asn Thr His Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln
Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85
90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys 100 105 110 Phe Gln Gly Ser His Ala Pro His Thr Phe Gly Gly Gly
Thr Lys Leu 115 120 125 Glu Leu Lys Arg 130 97393DNAMus musculus
97atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat
60gttttgatga cccaaaatcc actctccctg cctgtcagtc ttggagatca agcctccatc
120tcttgcagat ctagtcagag cattgtacat agtaatggaa acacccattt
agaatggtac 180ctgcagaaac caggccagtc tccaaagctc ctgatctaca
aagtttccaa ccgattttct 240ggggtcccag acaggttcag tggcagtgga
tcagggacag atttcacact caagatcagc 300agagtggagg ctgaggatct
gggagtttat tactgctttc aaggttcaca tgctccgcac 360acgttcggag
gggggaccaa gctggaactg aaa 39398143PRTMus musculus 98Met Lys Cys Ser
Trp Val Ile Phe Phe Leu Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn
Ser Glu Val Gln Leu Gln Gln Phe Gly Ala Glu Phe Val Arg 20 25 30
Ser Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35
40 45 Lys Asp Tyr Tyr Met Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly
Leu 50 55 60 Glu Trp Ile Gly Trp Ile Asp Pro Gly Asn Gly Asp Thr
Glu Cys Ala 65 70 75 80 Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala
Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu
Thr Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala Asp Arg
Tyr Asp Glu Gly Ala Ala Ser Asp Tyr 115 120 125 Ala Val Asp Tyr Trp
Gly Gln Gly Ser Ser Val Thr Val Ser Ser 130 135 140 99426DNAMus
musculus 99atgaaatgca gctgggtcat cttcttcctg atggcagtgg ttataggaat
caattcagag 60gttcagctgc agcagtttgg ggcagagttt gtgaggtcag gggcctcagt
caagttgtcc 120tgcacagctt ctggcttcaa cattaaagac tactatatgt
attgggtgaa acagaggcct 180gaacagggcc tggagtggat tggatggatt
gatcctggga atggtgatac tgaatgtgcc 240ccgaagttcc agggcaaggc
cactatgact gcagacacat cctccaacac agcctacctg 300cagctcagca
gcctgacatc tgaggacact gccgtctatt actgtaatgc cgatagatac
360gacgaggggg ccgcgtcgga ctatgctgtg gactactggg gtcaaggaag
ttcggtcaca 420gtctcg 426100132PRTMus musculus 100Met Lys Leu Pro
Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser
Ser Asp Val Leu Met Thr Gln Asn Pro Leu Ser Leu Pro Val 20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile 35
40 45 Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys
Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Glu Val Ser Asn
Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Gly Ala Glu
Asp Leu Gly Ile Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser Leu Phe Pro
His Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg 130
101393DNAMus musculus 101atgaagttgc ctgttaggct gttggtgctg
atgttctgga ttcctgcttc cagcagtgat 60gttttgatga cccaaaatcc actctccctg
cctgtcagtc ttggagatca agcctccatc 120tcttgcagat ctagtcagac
cattgtacat agtaatggaa acacctattt agaatggtac 180ctgcagaaac
caggccagtc tccaaagctc ctgatctacg aagtttccaa ccgattttct
240ggggtcccag acaggttcag tggcagtggc tcagggacag atttcacact
caaaatcagc 300agagtggggg ctgaagatct gggaatttat tactgctttc
aaggttcact ttttccgcac 360acgttcggag gggggaccaa gttggagctc aaa
393102143PRTMus musculus 102Met Glu Trp Ser Cys Ile Ile Phe Phe Leu
Met Ala Val Val Ile Gly 1 5 10 15 Ile Asn Ser Glu Val Gln Leu Gln
Gln Ser Gly Ala Glu Phe Val Arg 20 25 30 Ser Gly Ala Ser Val Lys
Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Tyr
Met Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp
Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Cys Ala 65 70 75 80
Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn 85
90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val 100 105 110 Tyr Tyr Cys Asn Ala Asp Arg Tyr Asp Glu Gly Ala Ala
Ser Asp Tyr 115 120 125 Ala Val Asp Tyr Trp Gly Gln Gly Ser Ser Val
Thr Val Ser Ser 130 135 140 103426DNAMus musculus 103atggaatgga
gctgtatcat cttcttcctg atggcagtgg ttataggaat caattcagag 60gttcagctgc
agcagtctgg ggcagagttt gtgaggtcag gggcctcagt caagttgtcc
120tgcacagctt ctggcttcaa cattaaagac tactatatgt attgggtgaa
acagaggcct 180gaacagggcc tggagtggat tggatggatt gatcctgaga
atggtgatac tgaatgtgcc 240ccgaagttcc agggcaaggc cactatgact
gcagacacat cctccaacac agcctacctg 300cagctcagca gcctgacatc
tgaggacact gccgtctatt actgtaatgc cgatagatac 360gacgaggggg
ccgcgtcgga ctatgctgtg gactactggg gtcaaggaag ctcagtcacc 420gtctcg
426104106PRTMus musculus 104Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85
90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 105327PRTMus
musculus 105Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115
120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235
240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu
Gly Lys 325 10613PRTOryctolagus cuniculus 106Gln Ser Ser Gln Arg
Val Phe Asn Asn Asn Glu Leu Ser 1 5 10 1077PRTOryctolagus cuniculus
107Glu Ala Ser Thr Leu Ala Ser 1 5 10811PRTOryctolagus cuniculus
108Leu Gly Gly Tyr Asp Asp Asp Gly Asp Asn Ala 1 5 10
1095PRTOryctolagus cuniculus 109Asn Tyr Tyr Met Thr 1 5
11016PRTOryctolagus cuniculus 110Leu Ile Tyr Pro Asp Gly Ser Thr
Phe Tyr
Ala Asn Trp Ala Glu Gly 1 5 10 15 11118PRTOryctolagus cuniculus
111Glu Gly Gly Ala Gly Asp Asn Thr Gly Thr Glu Tyr Tyr Tyr Gly Val
1 5 10 15 Asp Leu 11213PRTOryctolagus cuniculus 112Gln Ala Ser Glu
Asn Val Tyr Asp Lys Ser Ala Leu Ser 1 5 10 1137PRTOryctolagus
cuniculus 113Leu Ala Ser Thr Leu Ala Ser 1 5 11412PRTOryctolagus
cuniculus 114Ala Gly Tyr Lys Ser Ser Ile Ile Asp Gly Thr Ala 1 5 10
1155PRTOryctolagus cuniculus 115Asn Asn Ala Leu Ser 1 5
11616PRTOryctolagus cuniculus 116Ala Ile Gly Ala Gly Gly Asn Thr
Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 1177PRTOryctolagus
cuniculus 117Gly Asp Leu Pro Gly Gly Ile 1 5 11811PRTOryctolagus
cuniculus 118Gln Ala Ser Gln Ser Ile Ser Pro Ala Leu Ala 1 5 10
1197PRTOryctolagus cuniculus 119Asp Val Ser Lys Leu Ala Ser 1 5
12014PRTOryctolagus cuniculus 120Gln Ser Tyr Tyr Gly Ile Asn Ser
Asn Ser Tyr Gly Asn Ile 1 5 10 1215PRTOryctolagus cuniculus 121Ser
Tyr Ala Met Gly 1 5 12216PRTOryctolagus cuniculus 122Tyr Ile Tyr
Gly Asn Tyr Asn Lys Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
1236PRTOryctolagus cuniculus 123Gly Gly Ala Met Asp Val 1 5
12411PRTOryctolagus cuniculus 124Gln Ala Ser Glu Ser Ile Ser Thr
Trp Leu Ala 1 5 10 1257PRTOryctolagus cuniculus 125Arg Ala Ser Thr
Leu Ala Ser 1 5 12612PRTOryctolagus cuniculus 126Gln Gln Gly Trp
Asn Ile Asn Asn Ile Asp Asn Ile 1 5 10 1275PRTOryctolagus cuniculus
127Ser Tyr Ser Met Tyr 1 5 12816PRTOryctolagus cuniculus 128Phe Ile
Leu Ser Ala Thr Ala Val Ser Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
12913PRTOryctolagus cuniculus 129Asp Arg Asp Gly Gly Thr Thr Leu
Asp Gly Phe Asp Pro 1 5 10 13013PRTOryctolagus cuniculus 130Gln Ser
Ser Gln Ser Val Val Asn Asn Asn Asn Leu Ala 1 5 10
1317PRTOryctolagus cuniculus 131Phe Ala Ser Thr Leu Ala Ser 1 5
13212PRTOryctolagus cuniculus 132Gln Gly Thr Tyr Leu Ser Asp Asp
Trp Ser Asp Ala 1 5 10 1335PRTOryctolagus cuniculus 133Ser Ser Val
Met Asn 1 5 13416PRTOryctolagus cuniculus 134Ala Ile Trp Ser Gly
Gly Tyr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
13512PRTOryctolagus cuniculus 135Gly Gln Phe Gly Ala Ser Gly Gly
Gly Asp Val Leu 1 5 10 13613PRTOryctolagus cuniculus 136Gln Ser Ser
Pro Ser Val Asp Asn Asn Asn Leu Leu Ser 1 5 10 1377PRTOryctolagus
cuniculus 137Asp Ala Ser Thr Leu Ala Ser 1 5 13813PRTOryctolagus
cuniculus 138Val Ala Gly Tyr Gly Lys Arg Ser Arg Asp Ile Arg Val 1
5 10 1395PRTOryctolagus cuniculus 139Ser His Asn Met Gln 1 5
14016PRTOryctolagus cuniculus 140Ile Ile Tyr Pro Ser Asn Asn Ala
Tyr Tyr Ser Asn Trp Ala Lys Gly 1 5 10 15 1416PRTOryctolagus
cuniculus 141Asp Ile Asn Ser Ala Ile 1 5 14211PRTOryctolagus
cuniculus 142Gln Ala Ser Glu Ser Ile Tyr Ser Asn Leu Ala 1 5 10
1437PRTOryctolagus cuniculus 143Asp Ala Ser Asp Leu Ala Ser 1 5
14412PRTOryctolagus cuniculus 144Gln Cys Ser Trp Gly Gly Ser Thr
Tyr Gly Phe Ala 1 5 10 1455PRTOryctolagus cuniculus 145Ile Tyr Val
Met Thr 1 5 14616PRTOryctolagus cuniculus 146Ser Ile Asp Ala Asp
Asp Ser Ala Tyr Tyr Ala Thr Trp Ala Thr Ser 1 5 10 15
14711PRTOryctolagus cuniculus 147Gly Leu Tyr Ala Asn Gly Gly Pro
Phe Thr Leu 1 5 10 14813PRTOryctolagus cuniculus 148Gln Ser Ser Pro
Ser Val Tyr Asn Arg Asn Gln Leu Ser 1 5 10 1497PRTOryctolagus
cuniculus 149Thr Ala Ser Thr Leu Ala Ser 1 5 15012PRTOryctolagus
cuniculus 150Gln Gly Tyr Tyr Asn Thr Gly Ser Asp Thr Tyr Ala 1 5 10
1515PRTOryctolagus cuniculus 151Asn Tyr Asp Met Ser 1 5
15216PRTOryctolagus cuniculus 152Phe Ile Asn Thr Val Gly Tyr Ala
Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 1539PRTOryctolagus
cuniculus 153Leu Asp Asn Tyr Tyr Thr Trp Gly Ile 1 5
15411PRTOryctolagus cuniculus 154Gln Ala Ala Glu Asp Ile Tyr Ser
Ser Leu Ala 1 5 10 1557PRTOryctolagus cuniculus 155Ala Ala Ser Ile
Leu Ala Ser 1 5 15612PRTOryctolagus cuniculus 156Gln Thr Asn Tyr
Gly Ile Ser Ser Tyr Gly Ala Ala 1 5 10 1575PRTOryctolagus cuniculus
157Asn Lys Pro Ile Thr 1 5 15816PRTOryctolagus cuniculus 158Trp Ile
Ser Thr Thr Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
15910PRTOryctolagus cuniculus 159Tyr Ser Ser Asp Tyr Gly His His
Asp Leu 1 5 10 16012PRTOryctolagus cuniculus 160Gln Ala Ser Gln Ser
Val Tyr Arg Asp Tyr Leu Ser 1 5 10 1617PRTOryctolagus cuniculus
161Gly Ala Ser Asn Leu Ala Ser 1 5 16210PRTOryctolagus cuniculus
162Leu Gly Gly Phe Ser Gly Asn Ile Asn Thr 1 5 10
1635PRTOryctolagus cuniculus 163Asn Tyr His Met Cys 1 5
16416PRTOryctolagus cuniculus 164Ile Ile Ser Asn Thr Gly Tyr Thr
Tyr Tyr Ser Ser Trp Ala Lys Gly 1 5 10 15 1656PRTOryctolagus
cuniculus 165Asp Arg Leu Ala Asn Leu 1 5 16611PRTOryctolagus
cuniculus 166Gln Ala Ser Gln Ser Ile Ser Thr Tyr Leu Lys 1 5 10
1677PRTOryctolagus cuniculus 167Ser Ala Ser Thr Leu Thr Ser 1 5
16814PRTOryctolagus cuniculus 168Gln Ser Asn Ala Gly Ser Ser Ser
Ser Ser Cys Gly Tyr Ala 1 5 10 1695PRTOryctolagus cuniculus 169Tyr
Tyr Gly Met Gly 1 5 17016PRTOryctolagus cuniculus 170Ile Ile Ser
Gly Ile Gly Asn Thr Tyr Tyr Pro Thr Trp Ala Lys Gly 1 5 10 15
1713PRTOryctolagus cuniculus 171Gly Asp Phe 1 17211PRTOryctolagus
cuniculus 172Gln Ala Ser Gln Ser Ile Ser Arg Tyr Leu Lys 1 5 10
1737PRTOryctolagus cuniculus 173Ser Ala Ser Thr Leu Thr Ser 1 5
17414PRTOryctolagus cuniculus 174Gln Ser Asn Ala Gly Ser Ser Ser
Ser Ser Cys Gly Tyr Ala 1 5 10 1755PRTOryctolagus cuniculus 175Tyr
Tyr Gly Met Gly 1 5 17616PRTOryctolagus cuniculus 176Ile Ile Ser
Gly Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
1773PRTOryctolagus cuniculus 177Gly Asp Phe 1 17812PRTOryctolagus
cuniculus 178Gln Ser Ser Gln Ser Val Tyr Ser Asn Tyr Leu Ser 1 5 10
1797PRTOryctolagus cuniculus 179Val Ala Ser Ser Leu Ala Ser 1 5
18012PRTOryctolagus cuniculus 180Gly Gly Phe Gln Lys Tyr Ile Asp
Asp Gly Gly Ala 1 5 10 1815PRTOryctolagus cuniculus 181Thr Tyr Ala
Met Ser 1 5 18216PRTOryctolagus cuniculus 182Ile Met Ser Ser Ser
Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
1836PRTOryctolagus cuniculus 183Ser Ser Ser Phe Gly Leu 1 5
18412PRTMus musculus 184Ser Val Ser Ser Ser Ile Ser Ser Thr Asn Leu
His 1 5 10 1857PRTMus musculus 185Gly Thr Ser Asn Leu Ala Ser 1 5
1868PRTMus musculus 186Gln Gln Trp Ser Thr Thr Tyr Thr 1 5
1875PRTMus musculus 187Asp Tyr Tyr Ile His 1 5 18817PRTMus musculus
188Arg Ile Asp Pro Asp Asn Gly Glu Ser Thr Tyr Val Pro Lys Phe Gln
1 5 10 15 Gly 18913PRTMus musculus 189Glu Gly Leu Asp Tyr Gly Asp
Tyr Tyr Ala Val Asp Tyr 1 5 10 19015PRTMus musculus 190Arg Ala Ser
Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15
1917PRTMus musculus 191Arg Ala Ser Asn Leu Glu Ser 1 5 1929PRTMus
musculus 192Gln Gln Asn Tyr Glu Asp Pro Leu Thr 1 5 1935PRTMus
musculus 193Asn Tyr Leu Ile Glu 1 5 19417PRTMus musculus 194Val Ile
Asn Pro Gly Ser Gly Ile Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15
Ile 19517PRTMus musculus 195Asp Trp Asp Thr Phe Tyr Ser Tyr Glu Arg
Glu Val Tyr Ala Met Asp 1 5 10 15 Tyr 19611PRTMus musculus 196Arg
Ala Ser Gln Ser Ile Gly Thr Asn Ile His 1 5 10 1977PRTMus musculus
197Phe Ala Ser Glu Ser Ile Ser 1 5 1989PRTMus musculus 198Gln Gln
Ser Asn Ser Trp Pro Leu Thr 1 5 1995PRTMus musculus 199Asp Tyr Tyr
Val His 1 5 20017PRTMus musculus 200Trp Ile Asp Pro Glu Asn Gly Asp
Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly 2019PRTMus musculus
201Gly Tyr Gly Asn Phe Tyr Phe Asp Tyr 1 5 20211PRTMus musculus
202Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 2037PRTMus
musculus 203Trp Ala Ser Thr Arg His Thr 1 5 2049PRTMus musculus
204Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr 1 5 2055PRTMus musculus
205Asp Tyr Phe Met His 1 5 20617PRTMus musculus 206Trp Ile Asp Pro
Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly
2079PRTMus musculus 207Gly Gln Ala Thr Tyr Tyr Phe Asp Tyr 1 5
20811PRTMus musculus 208His Ala Thr Gln Asn Ile Asn Val Trp Leu Ser
1 5 10 2097PRTMus musculus 209Lys Thr Ser Asn Leu His Thr 1 5
2109PRTMus musculus 210Gln Gln Gly Gln Ser Phe Pro Tyr Thr 1 5
2115PRTMus musculus 211Asp Tyr Phe Met His 1 5 21217PRTMus musculus
212Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln
1 5 10 15 Gly 2139PRTMus musculus 213Gly Gln Ala Thr Tyr Tyr Phe
Asp Tyr 1 5 21417PRTMus musculus 214Lys Ser Ser Gln Ser Leu Leu Tyr
Ser Asn Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 2157PRTMus musculus
215Trp Ala Ser Thr Arg Glu Ser 1 5 2169PRTMus musculus 216Gln Gln
Tyr Tyr Asp Tyr Pro Trp Thr 1 5 2175PRTMus musculus 217Asp Tyr Phe
Met His 1 5 21817PRTMus musculus 218Trp Ile Asp Pro Glu Asn Gly Asp
Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly 2199PRTMus musculus
219Gly Gln Ala Thr Tyr Tyr Phe Asp Tyr 1 5 22011PRTMus musculus
220Arg Thr Ser Gln Asp Ile Asn Asn Phe Leu Asn 1 5 10 2217PRTMus
musculus 221His Thr Ser Arg Leu Lys Ser 1 5 2229PRTMus musculus
222Gln His Tyr Tyr Asn Leu Pro Trp Thr 1 5 2235PRTMus musculus
223Asp Asn Ile Met Val 1 5 22417PRTMus musculus 224Asn Ile Asn Pro
Tyr Tyr Gly Ser Pro Ser Tyr Asn Leu Lys Phe Lys 1 5 10 15 Asp
22510PRTMus musculus 225Glu Gly Gly Asn Tyr Gly Ser Leu Asp Asn 1 5
10 22617PRTMus musculus 226Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn
Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 2277PRTMus musculus 227Trp
Ala Ser Thr Arg Glu Ser 1 5 2289PRTMus musculus 228Gln Gln Tyr Tyr
Asp Tyr Pro Trp Thr 1 5 2295PRTMus musculus 229Asp Asn Ile Met Val
1 5 23017PRTMus musculus 230Asn Ile Asn Pro Tyr Tyr Gly Ser Pro Ser
Tyr Asn Leu Lys Phe Lys 1 5 10 15 Asp 23110PRTMus musculus 231Glu
Gly Gly Asn Tyr Gly Ser Leu Asp Asn 1 5 10 23211PRTMus musculus
232Arg Thr Ser Gln Asp Ile Asn Asn Phe Leu Asn 1 5 10 2337PRTMus
musculus 233His Thr Ser Arg Leu Lys Ser 1 5 2349PRTMus musculus
234Gln His Tyr Tyr Asn Leu Pro Trp Thr 1 5 2355PRTMus musculus
235Tyr Tyr Tyr Met Ser 1 5 23616PRTMus musculus 236Thr Ile Ser Ile
Asp Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Glu Gly 1 5 10 15
2379PRTMus musculus 237Gly His Ile Asn Thr Gly Met Asp Pro 1 5
23816PRTMus musculus 238Arg Ser Ser Gln Thr Ile Val His Ser Asn Gly
Asn Thr Tyr Leu Glu 1 5 10 15 2397PRTMus musculus 239Glu Val Ser
Asn Arg Phe Ser 1 5 2409PRTMus musculus 240Phe Gln Gly Ser His Phe
Pro His Thr 1 5 2415PRTMus musculus 241Asp Tyr Tyr Met Tyr 1 5
24217PRTMus musculus 242Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Cys
Ala Pro Lys Phe Gln 1 5 10 15 Gly 24315PRTMus musculus 243Asp Arg
Tyr Asp Glu Gly Ala Ala Ser Asp Tyr Gly Met Asp Tyr 1 5 10 15
24416PRTMus musculus 244Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly
Asn Thr His Leu Glu 1 5 10 15 2457PRTMus musculus 245Lys Val Ser
Asn Arg Phe Ser 1 5 2469PRTMus musculus 246Phe Gln Gly Ser His Ala
Pro His Thr 1 5 2475PRTMus musculus 247Asp Tyr Tyr Met Tyr 1 5
24817PRTMus musculus 248Trp Ile Asp Pro Gly Asn Gly Asp Thr Glu Cys
Ala Pro Lys Phe Gln 1 5 10 15 Gly 24915PRTMus musculus 249Asp Arg
Tyr Asp Glu Gly Ala Ala Ser Asp Tyr Ala Val Asp Tyr 1 5 10 15
25016PRTMus musculus 250Arg Ser Ser Gln Thr Ile Val His Ser Asn Gly
Asn Thr Tyr Leu Glu 1 5 10 15 2517PRTMus musculus 251Glu Val Ser
Asn Arg Phe Ser 1 5 2529PRTMus musculus 252Phe Gln Gly Ser Leu Phe
Pro His Thr 1 5 2535PRTMus musculus 253Asp Tyr Tyr Met Tyr 1 5
25417PRTMus musculus 254Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Cys
Ala Pro Lys Phe Gln 1 5 10 15 Gly 25515PRTMus musculus 255Asp Arg
Tyr Asp Glu Gly Ala Ala Ser Asp Tyr Ala Val Asp Tyr 1 5 10 15
256570DNAHomo sapiens 256caggggtggc aggcgttcaa gaatgatgcc
acggaaatca tccccgagct cggagagtac 60cccgagcctc caccggagct ggagaacaac
aagaccatga accgggcgga gaacggaggg 120cggcctcccc accacccctt
tgagaccaaa gacgtgtccg agtacagctg ccgcgagctg 180cacttcaccc
gctacgtgac cgatgggccg tgccgcagcg ccaagccggt caccgagctg
240gtgtgctccg gccagtgcgg cccggcgcgc ctgctgccca acgccatcgg
ccgcggcaag 300tggtggcgac ctagtgggcc cgacttccgc tgcatccccg
accgctaccg cgcgcagcgc 360gtgcagctgc tgtgtcccgg tggtgaggcg
ccgcgcgcgc gcaaggtgcg cctggtggcc 420tcgtgcaagt gcaagcgcct
cacccgcttc cacaaccagt cggagctcaa ggacttcggg 480accgaggccg
ctcggccgca gaagggccgg aagccgcggc cccgcgcccg gagcgccaaa
540gccaaccagg ccgagctgga gaacgcctac 570
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